Modification of feeding behaviour

ABSTRACT

Methods are disclosed for decreasing calorie intake, food intake, and appetite in a subject. The methods include peripherally administering PYY or an agonist thereof and GLP-1 or an agonist thereof to the subject, simultaneously or sequentially, thereby decreasing the calorie intake of the subject.

STATEMENT OF GOVERNMENT SUPPORT

This disclosure was made in part with United States government supportpursuant to grants RR00163, DK51730 and DK55819, from the NationalInstitutes of Health. The United States government has certain rights inthe disclosure.

PRIORITY CLAIM

This application claims the benefit of UK Application No. GB0200507.2filed Jan. 10, 2002, and International Application PCT/US02/31944 whichare both incorporated by reference in their entirety herein.

FIELD

This application relates to the use of agents to control appetite,feeding, food intake, energy expenditure and calorie intake,particularly in the field of obesity.

BACKGROUND

According to the National Health and Nutrition Examination Survey(NHANES III, 1988 to 1994), between one third and one half of men andwomen in the United States are overweight. In the United States, sixtypercent of men and fifty-one percent of women, of the age of 20 orolder, are either overweight or obese. In addition, a large percentageof children in the United States are overweight or obese.

The cause of obesity is complex and multi-factorial. Increasing evidencesuggests that obesity is not a simple problem of self-control but is acomplex disorder involving appetite regulation and energy metabolism. Inaddition, obesity is associated with a variety of conditions associatedwith increased morbidity and mortality in a population. Although theetiology of obesity is not definitively established, genetic, metabolic,biochemical, cultural and psychosocial factors are believed tocontribute. In general, obesity has been described as a condition inwhich excess body fat puts an individual at a health risk.

There is strong evidence that obesity is associated with increasedmorbidity and mortality. Disease risk, such as cardiovascular diseaserisk and type 2 diabetes disease risk, increases independently withincreased body mass index (BMI). Indeed, this risk has been quantifiedas a five percent increase in the risk of cardiac disease for females,and a seven percent increase in the risk of cardiac disease for males,for each point of a BMI greater than 24.9 (see Kenchaiah et al., N.Engl. J. Med. 347:305, 2002; Massie, N. Engl. J. Med. 347:358, 2002). Inaddition, there is substantial evidence that weight loss in obesepersons reduces important disease risk factors. Even a small weightloss, such as 10% of the initial body weight in both overweight andobese adults has been associated with a decrease in risk factors such ashypertension, hyperlipidemia, and hyperglycemia.

Although diet and exercise provide a simple process to decrease weightgain, overweight and obese individuals often cannot sufficiently controlthese factors to effectively lose weight. Pharmacotherapy is available;several weight loss drugs have been approved by the Food and DrugAdministration that can be used as part of a comprehensive weight lossprogram. However, many of these drugs have serious adverse side effects.When less invasive methods have failed, and the patient is at high riskfor obesity related morbidity or mortality, weight loss surgery is anoption in carefully selected patients with clinically severe obesity.However, these treatments are high-risk, and suitable for use in only alimited number of patients. It is not only obese subjects who wish tolose weight. People with weight within the recommended range, forexample, in the upper part of the recommended range, may wish to reducetheir weight, to bring it closer to the ideal weight. Thus, a needremains for agents that can be used to effect weight loss in overweightand obese subjects.

SUMMARY

Disclosed herein are findings that peripheral administration of PYY, oran agonist thereof, to a subject results in decreased food intake,caloric intake, and appetite, and an alteration in energy metabolism.The subject can be any subject, including, but not limited to, a humansubject. In several embodiments, the subject desires to lose weight, isobese, overweight, or suffers from a weight-related disorder. PYY₃₋₃₆can preferably be administered to the subject.

Also disclosed are findings that co-administration of PYY, or an agonistthereof, for example, PYY₃₋₃₆, with GLP-1 or an agonist thereof to asubject results in a greater decrease in food intake, caloric intake,and appetite, than when PYY or an agonist thereof is administered alone.The effect is synergistic.

In one embodiment, a method is disclosed for decreasing calorie intakein a subject. The method includes peripherally administering atherapeutically effective amount of PYY or an agonist thereof and GLP-1or an agonist thereof to the subject, thereby decreasing the calorieintake of the subject.

In another embodiment, a method is disclosed for decreasing appetite ina subject. The method includes peripherally administering atherapeutically effective amount of PYY or an agonist thereof and GLP-1or an agonist thereof to the subject, thereby decreasing the appetite ofthe subject.

In a further embodiment, a method is disclosed for decreasing foodintake in a subject. The method includes peripherally administering atherapeutically effective amount of PYY or an agonist thereof, and GLP-1or an agonist thereof to the subject, thereby decreasing the food intakeof the subject.

In yet another embodiment, a method is disclosed herein for increasingenergy expenditure in a subject. The method includes peripherallyadministering a therapeutically effective amount of PYY or an agonistthereof, and GLP-1 or an agonist thereof to the subject, therebyincreasing energy expenditure in the subject.

A method is also disclosed for decreasing calorie intake, food intake,or appetite in a human subject. The method includes peripherallyinjecting a therapeutically effective amount of PYY or an agonistthereof in a pharmaceutically acceptable carrier to the subject in apulse dose, thereby decreasing the calorie intake, food intake, orappetite of the subject, and also administering GLP-1 or an agonistthereof.

In each method above, the GLP-1 or an agonist thereof may beadministered simultaneously or substantially simultaneously as the PYYor agonist thereof, or sequentially, in any order. The PYY or agonistthereof and the GLP-1 or agonist thereof may be administered in a singlepharmaceutical composition or in separate compositions, and they may beadministered by the same route or my different routes.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of diagrams and digital images showing the generation oftransgenic mice expressing EGFP in ARC POMC neurons. FIG. 1 a is aschematic diagram of the structure of the POMC-EGFP transgene. FIG. 1 bis a digital image showing the identification of a single POMC neuron(arrowhead on recording electrode tip) by EGFP fluorescence (upper) andIR-DIC microscopy (lower) in a living ARC slice prior toelectrophysiological recordings. FIG. 1 c is a set of digital imagesshowing the co-localization (bright, on right) of EGFP (left) andβ-endorphin immunoreactivity (middle) in ARC POMC neurons. Scale bars: b& c, 50 μm. FIG. 1 d is a set of diagrams showing the distribution ofEGFP-positive neuronal soma throughout the ARC nucleus. ∘=5 cells, •=10cells.

FIG. 2 is a tracing and graphs showing activation of MOP-Rshyperpolarizes the EGFP-labeled POMC neurons by opening Gprotein-coupled inwardly-rectifying potassium channels. FIG. 2 a is atracing showing met-enkephalin hyperpolarizes POMC neurons and inhibitsall action potentials. The horizontal bar indicates the time when 30 μMMet-Enk was bath-applied to the slice. FIG. 2 b is a graph showingmet-enkephalin current and reversal potential is shifted byextracellular K⁺ concentration. FIG. 2 c is a graph showingmet-enkephalin activates MOP-R5 on POMC neurons. A Met-Enk (30 μM)current was observed and the MOP-R specific antagonist CTAP (1 μM) wasapplied for 1 minute. Following CTAP Met-Enk elicited no current. Thefigure is representative of three experiments.

FIG. 3 are tracings and graphs demonstrating that leptin depolarizesPOMC neurons via a non-specific cation channel, and decreases GABAergictone onto POMC cells. FIG. 3 a is a tracing demonstrating that leptindepolarizes POMC neurons and increases the frequency of actionpotentials within 1 to 10 minutes of addition. The figure is arepresentative example of recordings made from 77 POMC neurons. FIG. 3 bis a graph showing that leptin causes a concentration dependentdepolarization of POMC cells. The depolarization caused by leptin wasdetermined at 0.1, 1, 10, 50, and 100 nM (EC₅₀=5.9 nM) in (8, 7, 9, 3,45) cells respectively. FIG. 3 c is a graph showing that leptindepolarizes POMC cells by activating a nonspecific cation current. Thefigure is representative of the response in 10 cells. FIG. 3 d is agraph showing that leptin decreases the frequency of IPSCs in POMCcells. The figure is an example of 5 cells in which leptin (100 nM)decreased the frequency of IPSCs. FIG. 3 e is a tracing demonstratingthat leptin had no effect on 5 adjacent non-fluorescent ARC neurons.FIG. 3 f is a tracing showing that leptin hyperpolarized 5non-fluorescent ARC neurons.

FIG. 4 is a set of images showing that the GABAergic inputs to POMCcells are from NPY neurons that co-express GABA. FIG. 4 a is a graphshowing that NPY decreases the frequency of mini IPSCs in POMC neurons.FIG. 4 b is a graph demonstrating that D-Trp⁸-γMSH (7 nM), a dose thatselectively activates MC3-R, increases the frequency of GABAergic IPSCsin POMC neurons. FIG. 4 c is a tracing showing that D-Trp⁸-γMSHhyperpolarizes POMC neurons. FIGS. 4 a, 4 b and 4 c are representative.FIG. 4 d is a set of digital images demonstrating that expression of NPYin nerve terminals adjacent to POMC neurons in the ARC. NPY nerveterminals (black, arrowheads); POMC neuronal soma (grey). Scale bar, 10μm. FIG. 4 e is a digital image showing expression of GABA and NPY innerve terminals synapsing onto POMC neurons in the ARC. GABAimmunoreactivity (10 nm gold particles, arrowheads without tail) and NPYimmunoreactivity (25 nm gold particles, arrows with tail) are inseparate vesicle populations co-localized within synaptic boutons thatmake direct contact with the soma of POMC neurons (DAB contrasted withuranyl acetate and lead citrate, diffuse black in cytoplasm). Scale bar,1 μm. FIG. 4 f is a diagram of the model of NPY/GABA and POMC neurons inthe ARC.

FIG. 5 is a set of graphs relating to the feeding response to PYY₃₋₃₆ inrats. FIG. 5 a is a bar graph of dark-phase feeding tabulating foodintake after intraperitoneal injection of PYY₃₋₃₆. Freely feeding ratswere injected with PYY₃₋₃₆ at the doses indicated (μg/100 g), or saline,just prior to ‘lights off’ and 4-hour cumulative food intake wasmeasured. Results are the mean±s.e.m. (n=8 per group), *=p<0.05,**=p<0.01, ***=<0.001 compared to saline. FIG. 5 b is a bar graph offood intake after intraperitoneal injection of PYY₃₋₃₆. Fasted rats wereinjected with PYY₃₋₃₆ at the doses indicated (μg/100 g), or saline, and4-hour cumulative food intake was measured. Results are shown as themean±s.e.m. (n=8 per group), *=p<0.05, **=p<0.01, ***=<0.001 compared tosaline. FIG. 5 c is a bar graph of cumulative food intake afterintraperitoneal injection of saline or PYY₃₋₃₆. Fasted rats wereinjected with either saline (closed bars) or PYY₃₋₃₆ 5 μg/100 g (openbars) and cumulative food intake measured at the time points indicated.Results are expressed as mean±s.e.m. (n=12 per group), **=p<0.01compared to saline. FIG. 5 d is a line graph of body weight gain duringchronic treatment with PYY₃₋₃₆. Rats were injected intraperitoneallywith PYY₃₋₃₆ 5 μg/100 g (open squares) or saline (filled invertedtriangles) twice daily for 7 days. Body weight gain was calculated eachday. Results are expressed as mean±s.e.m. (n=12 per group)**=p<0.01compared to saline.

FIG. 6 is a set of digital images of c-fos expression in Pomc-EGFP mice.FIGS. 6 a and 6 b are digital images of representative sections (bregma−1.4 mm²²) of c-fos expression in the arcuate nucleus of Pomc-EGFP miceresponse to intraperitoneal saline (FIG. 6 a) or PYY₃₋₃₆ (5 μg/100 g)(FIG. 6 b). Scale bar 100 μm. 3V, third ventricle; Arc, arcuate nucleus.FIGS. 6 c and 6 d are digital images of representative sections showingPOMC-EGFP neurons (FIG. 6 c) and c-fos immunoreactivity (FIG. 6 d)either co-localizing (bright arrows) or alone (single darker arrow).Scale bar 25 μm.

FIG. 7 is a set of bar graphs relating to intra-arcuate PYY₃₋₃₆ in ratsand feeding effects of IP PYY₃₋₃₆ in Y2r-null mice. FIG. 7 a is a bargraph of food intake following intra-arcuate PYY₃₋₃₆ injection. Fastedrats were injected with saline or PYY₃₋₃₆ into the arcuate nucleus atthe doses indicated. Post-injection 2-hour food intake was measured,**=p<0.01 compared to saline. FIGS. 7 b and 7 c are bar graphs offeeding response to PYY₃₋₃₆ in Y2r-null mice following IPadministration: wild type littermates mice (FIG. 7 b) and Y2r-null mice(FIG. 7 c), fasted for 24 hours, were injected with PYY₃₋₃₆ at the dosesindicated (μg/100 g), or saline, and 4-hour cumulative food intake wasmeasured. Results are the mean±s.e.m. (n=5 per group), *=p<0.05,**=p<0.01 compared to saline.

FIG. 8 is a set of images relating to the electrophysiological andneuropeptide responses to PYY₃₋₃₆ and Y2A. FIG. 8 a is a tracing showingthe effect of PYY₃₋₃₆ (10 nM) on the frequency of action potentials inPOMC neurons (whole-cell configuration recordings; n=22)*p<0.05. PYY₃₋₃₈was administered at time D for 3 minutes; baseline, −3 to 0 minute;PYY₃₋₃₆, 2-5 minutes; and wash-out, 8-11 minutes. Inset shows arepresentative recording of membrane potential and action potentialfrequency. FIG. 8 b is a graph of the effect if PYY₃₋₃₈ (10 nM) on thefrequency of action potentials in loose cell-attached patch recordings(n=8). Data from individual cells were normalized to the firing rate forthe 200 s before PYY₃₋₃₈ addition. FIG. 8 c is a tracing and a graph ofthe effect of PYY₃₋₃₆ (50 nM) on spontaneous IPSCs onto POMC neurons(n=13). Inset shows a representative recording of IPSCs before and afterPYY₃₋₃₆ (50 nM), respectively. Results in FIG. 8 a-8 c are expressed asmean∀s.e.m. FIGS. 8 d and 8 e are bar graphs showing NPY (FIG. 8 d) and%-MSH (FIG. 8 e) released from hypothalamic explants in response to Y2A.Hypothalamic slices were incubated with artificial CSF (aCSF), with orwithout 50 nM Y2A, for 45 minutes. Results are expressed as mean∀s.e.m.(n=40); **=p<0.01; ***=p<0.001 compared to saline.

FIG. 9 is a set of graphs showing the effect of PYY₃₋₃₆ infusion onappetite and food intake in human subjects. FIG. 9 a is a graph of thecalorie intake from a “free-choice” buffet meal 2 hours after infusionwith saline or PYY₃₋₃₆. The thin lines indicate individual changes incalorie intake for each subject between saline and PYY₃₋₃₆administration. The thick line represents mean change between the twoinfusions (n=12). FIG. 9 b is a graph of the 24-hour calorie intakefollowing infusion with saline or PYY₃₋₃₆. Total calorie intake, asassessed by food diaries, is shown for the 24-hour period followingeither saline or PYY₃₋₃₆ infusion. Data is given as mean±s.e.m. (n=12),***=p<0.0001 compared to saline. FIG. 9 c is a graph of the appetitescore (relative scale). Visual analogue scores (Raben et al., Br. J.Nutr. 73, 517-30, 1995) show perceived hunger during and afterinfusions. The results are presented as change from baseline scores andare the mean±s.e.m. for all 12 subjects.

FIG. 10 shows plasma PYY₃₋₃₆ levels following subcutaneousadministration of 10 nmols PYY₃₋₃₆ (broken line) and 20 nmols PYY₃₋₃₆(solid line).

FIG. 11 shows the effect on 1-hour food intake in non-fasted rats of IPadministration of GLP-1 in the presence or absence of concomitantexendin 9-39 in the arcuate nucleus of the rat. S=Saline, G=GLP-1 (25nmol/kg), and Ex=exendin 9-39 (20 nmoles/kg).

FIG. 12 shows the effects of co-administration of PYY₃₋₃₆ and GLP-1 on1-hour food intake in non-fasted rats injected (intraperitoneally) priorto the onset of the dark phase. IP GLP-1 (30 μg/kg=1×G or 60 μg/kg=2×Gin 500 μl saline) or IP PYY₃₋₃₆ (3 μg/kg=1×P or 6 μg/kg=2×P in 500 μlsaline). Co-administration (P+G group) is GLP-1 30 μg/kg and PYY₃₋₃₆3μg/kg combined.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand.

DETAILED DESCRIPTION I. Abbreviations

-   -   α-MSH: alpha melanocortin stimulating hormone    -   Arc: arcuate nucleus    -   EPSP: excitatory postsynaptic potential    -   GABA: γaminobutyric acid    -   GFP, EGFP: green fluorescent protein    -   GLP-1: glucagons-like peptide-1    -   ICV: intracerebroventricular    -   IP: intraperitoneal    -   IPSC: inhibitory postsynaptic current    -   kb: kilobase    -   kg: kilogram    -   MOP-R: μ-opiod receptor    -   MV: millivolts    -   NPY: neuropeptide Y    -   Oxm: oxyntomodulin    -   pmol: picomole    -   POMC: proopiomelanocortin    -   RIA: radioimmunoassay    -   RPA: RNase protection assay    -   s.e.m: standard error of the mean    -   TH: tyrosine hydroxylase    -   μM: micromolar    -   V: volts    -   Y2A: N-acetyl (Leu²⁸, Leu³¹) NPY (24-36)

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Action potential: A rapidly propagated electrical message that speedsalong an axon of a neuron and over the surface membrane of many muscleand glandular cells. In axons they are brief, travel at constantvelocity, and maintain a constant amplitude. Like all electricalmessages of the central nervous system, the action potential is amembrane potential change caused by the flow of ions through ionchannels in the membrane. In one embodiment, an action potential is aregenerative wave of sodium permeability.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Anorexia: A lack or loss of the appetite for food. In one embodiment,anorexia is a result of “anorexia nervosa.” This is an eating disorderprimarily affecting females, usually with onset in adolescence,characterized by refusal to maintain a normal minimal body weight,intense fear of gaining weight or becoming obese, and a disturbance ofbody image resulting in a feeling of being fat or having fat in certainareas even when extremely emaciated, undue reliance on body weight orshape for self-evaluation, and amenorrhea. Associated features ofteninclude denial of the illness and resistance to psychotherapy,depressive symptoms, markedly decreased libido, and obsessions orpeculiar behavior regarding food, such as hoarding. The disorder isdivided into two subtypes, a restricting type, in which weight loss isachieved primarily through diet or exercise, and a binge-eating/purgingtype, in which binge eating or purging behavior also occur regularly.

Antagonist: A substance that tends to nullify the action of another, asan agent that binds to a cell receptor without eliciting a biologicalresponse, blocking binding of substances that could elicit suchresponses.

Appetite: A natural desire, or longing for food. In one embodiment,appetite is measured by a survey to assess the desire for food.Increased appetite generally leads to increased feeding behavior.

Appetite Suppressants: Compounds that decrease the desire for food.Commercially available appetite suppressants include, but are notlimited to, amfepramone (diethylpropion), phentermine, mazindol andphenylpropanolamine fenfluramine, dexfenfluramine, and fluoxetine.

Binding: A specific interaction between two molecules, such that the twomolecules interact. Binding can be specific and selective, so that onemolecule is bound preferentially when compared to another molecule. Inone embodiment, specific binding is identified by a disassociationconstant (K_(d)).

Body Mass Index (BMI): A mathematical formula for measuring body mass,also sometimes called Quetelet's Index. BMI is calculated by dividingweight (in kg) by height² (in meters). The current standards for bothmen and women accepted as “normal” are a BMI of 20-24.9 kg/m². In oneembodiment, a BMI of greater than 25 kg/m² can be used to identify anobese subject. Grade I obesity corresponds to a BMI of 25-29.9 kg/m².Grade II obesity corresponds to a BMI of 30-40 kg/m²; and Grade IIIobesity corresponds to a BMI greater than 40 kg/m² (Jequier, Am. J Clin.Nutr. 45:1035-47, 1987). Ideal body weight will vary among species andindividuals based on height, body build, bone structure, and sex.

c-fos: The cellular homologue of the viral v-fos oncogene found in FBJ(Finkel-Biskis-Jinkins) and FBR murine osteosarcoma viruses (MSV). Thehuman fos gene maps to chromosome 14q21-q31. Human fos has beenidentified as TIS-28.

C-fos is thought to have an important role in signal transduction, cellproliferation, and differentiation. It is a nuclear protein which, incombination with other transcription factors (for example, jun) acts asa trans-activating regulator of gene expression. C-fos is an immediateearly response gene, which are believed to play a key role in the earlyresponse of cells to growth factors. C-fos is involved also in thecontrol of cell growth and differentiation of embryonic hematopoieticcells and neuronal cells. The human c-fos coding amino acid and nucleicsequences are known (e.g., see Verma et al., Cold Spring Harb. Symp.Quant. Biol. 51, 949, 1986; GenBank Accession Nos. K00650 and M16287,and is available on the internet).

Caloric intake or calorie intake: The number of calories (energy)consumed by an individual.

Calorie: A unit of measurement in food. A standard calorie is defined as4.184 absolute joules, or the amount of energy it takes to raise thetemperature of one gram of water from 15 to 16° C. (or 1/100th theamount of energy needed to raise the temperature of one gram of water atone atmosphere pressure from 0° C. to 100° C.), food calories areactually equal to 1,000 standard calories (1 food calorie=1kilocalorie).

Conservative variation: The replacement of an amino acid residue byanother, biologically similar residue. Examples of conservativevariations include the substitution of one hydrophobic residue such asisoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as the substitutionof arginine for lysine, glutamic for aspartic acid, or glutamine forasparagine, and the like. The term “conservative variation” alsoincludes the use of a substituted amino acid in place of anunsubstituted parent amino acid provided that antibodies raised to thesubstituted polypeptide also immunoreact with the unsubstitutedpolypeptide.

Non-limiting examples of conservative amino acid substitutions includethose listed below:

Original Conservative Residue Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Depolarization: An increase in the membrane potential of a cell. Certainstimuli reduce the charge across the plasma membrane. These can beelectrical stimuli (which open voltage-gated channels), mechanicalstimuli (which activate mechanically-gated channels) or certainneurotransmitters (which open ligand-gated channels). In each case, thefacilitated diffusion of sodium into the cell increases the restingpotential at that spot on the cell creating an excitatory postsynapticpotential (EPSP). Depolarizations can also be generated by decreasingthe frequency of inhibitory postsynaptic currents (IPSCs), these are dueto inhibitory neurotransmitters facilitating the influx of chloride ionsinto the cell, creating an IPSC. If the potential is increased to thethreshold voltage (about −50 mV in mammalian neurons), an actionpotential is generated in the cell.

Diabetes: A failure of cells to transport endogenous glucose acrosstheir membranes either because of an endogenous deficiency of insulinand/or a defect in insulin sensitivity. Diabetes is a chronic syndromeof impaired carbohydrate, protein, and fat metabolism owing toinsufficient secretion of insulin or to target tissue insulinresistance. It occurs in two major forms: insulin-dependent diabetesmellitus (IDDM, type I) and non-insulin dependent diabetes mellitus(NIDDM, type II) which differ in etiology, pathology, genetics, age ofonset, and treatment.

The two major forms of diabetes are both characterized by an inabilityto deliver insulin in an amount and with the precise timing that isneeded for control of glucose homeostasis. Diabetes type I, or insulindependent diabetes mellitus (IDDM) is caused by the destruction of βcells, which results in insufficient levels of endogenous insulin.Diabetes type II, or non-insulin dependent diabetes, results from adefect in both the body's sensitivity to insulin, and a relativedeficiency in insulin production.

Exendin: A 39-amino acid peptide isolated from the salivary glands ofthe Gila monster (Heloderma suspectum) (Eng J et al J Biol Chem267:7402-7405, 1992), see SEQ ID NO:341. Exendin is an example of anagonist at the GLP-1 receptor. Molecules derived from exendin-4 and thatalso have GLP-1 agonist activity are further examples of GLP-1 agonists.

Food intake: The amount of food consumed by an individual. Food intakecan be measured by volume or by weight. In one embodiment, food intakeis the total amount of food consumed by an individual. In anotherembodiment, food intake is the amount of proteins, fat, carbohydrates,cholesterol, vitamins, minerals, or any other food component, of theindividual. “Protein intake” refers to the amount of protein consumed byan individual. Similarly, “fat intake,” “carbohydrate intake,”“cholesterol intake,” “vitamin intake,” and “mineral intake” refer tothe amount of proteins, fat, carbohydrates, cholesterol, vitamins, orminerals consumed by an individual.

Glucagon-like peptide-1: Peptides produced by processing ofpreproglucogon, which is a 160 amino acid polypeptide, in the centralnervous system (CNS) and the intestine. GLP-1 (1-37) is the initialproduct. GLP-1 (1-37) is amidated by post-translational processing toyield GLP-1 (1-36) NH, or is enzymatically processed to give GLP-1(7-37) (SEQ ID NO: 338). GLP-1 (7-37) can be amidated to give GLP-1(7-36) amide (SEQ ID NO: 339), which is the most biologically activeform. GLP-1 is released into the circulation in response to nutrientintake. Intestinal cells secrete GLP-1 (7-37) and GLP-1 (7-36) amide ina ratio of 1 to 5.

GLP-1 receptors are found in the brainstem, arcuate nucleus andparaventricular nucleus. Physiological actions of GLP-1 in man includestimulation of insulin release, suppression of gastric acid secretionand slowing of gastric emptying.

A GLP-1 agonist is a peptide, small molecule, or chemical compound thatpreferentially binds to the GLP-1 receptor and stimulate the samebiological activity as GLP-1. In one embodiment, an agonist for theGLP-1 receptor binds to the receptor with an equal or greater affinitythan a GLP-1 peptide. In another embodiment, an agonist selectivelybinds the GLP-1 receptor, as compared to binding to another receptor.

One of skill in the art can readily determine the dissociation constant(K_(d)) value of a given compound. This value is dependent on theselectivity of the compound tested. For example, a compound with a K_(d)which is less than 10 nM is generally considered an excellent drugcandidate. However, a compound that has a lower affinity, but isselective for the particular receptor, can also be a good drugcandidate. In one specific, non-limiting example, an assay, such as acompetition assay, is used to determine if a compound of interest is aGLP-1 receptor agonist.

GLP-1 agonists include GLP-1 related peptides and peptides that resultfrom natural or synthetic enzymatic or chemical processing of a GLP-1peptide or a related peptide.

Hyperpolarization: A decrease in the membrane potential of a cell.Inhibitory neurotransmitters inhibit the transmission of nerve impulsesvia hyperpolarization. This hyperpolarization is called an inhibitorypostsynaptic potential (IPSP). Although the threshold voltage of thecell is unchanged, a hyperpolarized cell requires a stronger excitatorystimulus to reach threshold.

Inhibitory Postsynaptic Current: A current that inhibits anelectrophysiological parameter of a postsynaptic cell. The potential ofa postsynaptic cell can be analyzed to determine an effect on apresynaptic cell. In one embodiment, the postsynaptic cell is held involtage clamp mode, and postsynaptic currents are recorded. Ifnecessary, antagonists of other classes of current can be added. In onespecific, non-limiting example, to record GABAergic IPSCs, blockers ofexcitatory channels or receptors can be added. The instantaneousfrequency over time is then determined.

In one embodiment, IPSCs give a measure of the frequency of GABA releasefrom an NPY neuron. Thus, as NPY neurons release GABA onto POMC neurons,measurement of IPSC frequency is a gauge of the inhibitory tone thatPOMC neurons are receiving, and can be used to assess the effect of anagonist of PYY.

Membrane potential: The electrical potential of the interior of the cellwith respect to the environment, such as an external bath solution. Oneof skill in the art can readily assess the membrane potential of a cell,such as by using conventional whole cell techniques. Activation of acell is associated with less negative membrane potentials (for exampleshifts from about −50 mV to about −40 mV). These changes in potentialincrease the likelihood of action potentials, and thus lead to anincrease in the rate of action potentials.

The rate of action potentials can be assessed using many approaches,such as using conventional whole cell access, or using, for example,perforated-patch whole-cell and cell-attached configurations. In eachevent the absolute voltage or current is not assessed, rather thefrequency of rapid deflections characteristic of action potentials isassessed, as a function of time (therefore this frequency is aninstantaneous frequency, reported in “bins”). This time component can berelated to the time at which a compound, such as a PYY agonist, isapplied to the bath to analyze the effect of the compound, such as thePYY agonist, on action potential firing rate.

Neuropeptide Y (NPY): A 36-amino acid peptide that is a neuropeptideidentified in the mammalian brain. NPY is believed to be an importantregulator in both the central and peripheral nervous systems andinfluences a diverse range of physiological parameters, includingeffects on psychomotor activity, food intake, central endocrinesecretion, and vasoactivity in the cardiovascular system. Highconcentrations of NPY are found in the sympathetic nerves supplying thecoronary, cerebral, and renal vasculature and have contributed tovasoconstriction. NPY binding sites have been identified in a variety oftissues, including spleen, intestinal membranes, brain, aortic smoothmuscle, kidney, testis, and placenta. In addition, binding sites havebeen reported in a number of rat and human cell lines.

Neuropeptide Y (NPY) receptor has structure/activity relationshipswithin the pancreatic polypeptide family. This family includes NPY,which is synthesized primarily in neurons; peptide YY (PYY), which issynthesized primarily by endocrine cells in the gut; and pancreaticpolypeptide (PP), which is synthesized primarily by endocrine cells inthe pancreas. These 36 amino acid peptides have a compact helicalstructure involving an amino acid structure, termed a “PP-fold” in themiddle of the peptide.

NPY binds to several receptors, including the Y1, Y2, Y3, Y4 (PP), Y5,Y6, and Y7 receptors. These receptors are recognized based on bindingaffinities, pharmacology, and sequence (if known). Most, if not all ofthese receptors are G protein coupled receptors. The Y1 receptor isgenerally considered to be postsynaptic and mediates many of the knownactions of neuropeptide Y in the periphery. Originally, this receptorwas described as having poor affinity for C-terminal fragments ofneuropeptide Y, such as the 13-36 fragment, but interacts with the fulllength neuropeptide Y and peptide YY with equal affinity (e.g., see PCTpublication WO 93/09227).

Pharmacologically, the Y2 receptor is distinguished from Y1 byexhibiting affinity for C-terminal fragments of neuropeptide Y. The Y2receptor is most often differentiated by the affinity of neuropeptideY(13-36), although the 3-36 fragment of neuropeptide Y and peptide YYprovides improved affinity and selectivity (see Dumont et al., Societyfor Neuroscience Abstracts 19:726, 1993). Signal transmission throughboth the Y1 and the Y2 receptors are coupled to the inhibition ofadenylate cyclase. Binding to the Y-2 receptor was also found to reducethe intracellular levels of calcium in the synapse by selectiveinhibition of N-type calcium channels. In addition, the Y-2 receptor,like the Y1 receptors, exhibits differential coupling to secondmessengers (see U.S. Pat. No. 6,355,478). Y2 receptors are found in avariety of brain regions, including the hippocampus, substantianigra-lateralis, thalamus, hypothalamus, and brainstem. The human,murine, monkey and rat Y2 receptors have been cloned (e.g., see U.S.Pat. No. 6,420,352 and U.S. Pat. No. 6,355,478).

A Y2 receptor agonist is a peptide, small molecule, or chemical compoundthat preferentially binds to the Y2 receptor and stimulatesintracellular signaling. In one embodiment, an agonist for the Y2receptor binds to the receptor with an equal or greater affinity thanNPY. In another embodiment, an agonist selectively binds the Y2receptor, as compared to binding to another receptor.

One of skill in the art can readily determine the dissociation constant(K_(d)) value of a given compound. This value is dependent on theselectivity of the compound tested. For example, a compound with a K_(d)which is less than 10 nM is generally considered an excellent drugcandidate. However, a compound that has a lower affinity, but isselective for the particular receptor, can also be a good drugcandidate. In one specific, non-limiting example, an assay, such as acompetition assay, is used to determine if a compound of interest is aY2 receptor agonist. Assays useful for evaluating neuropeptide Yreceptor antagonists are also well known in the art (see U.S. Pat. No.5,284,839, which is herein incorporated by reference, and Walker et al.,Journal of Neurosciences 8:2438-2446, 1988).

Normal Daily Diet: The average food intake for an individual of a givenspecies. A normal daily diet can be expressed in terms of caloricintake, protein intake, carbohydrate intake, and/or fat intake. A normaldaily diet in humans generally comprises the following: about 2,000,about 2,400, or about 2,800 to significantly more calories. In addition,a normal daily diet in humans generally includes about 12 g to about 45g of protein, about 120 g to about 610 g of carbohydrate, and about 11 gto about 90 g of fat. A low calorie diet would be no more than about85%, and preferably no more than about 70%, of the normal caloric intakeof a human individual.

In animals, the caloric and nutrient requirements vary depending on thespecies and size of the animal. For example, in cats, the total caloricintake per pound, as well as the percent distribution of protein,carbohydrate and fat varies with the age of the cat and the reproductivestate. A general guideline for cats, however, is 40 cal/lb/day (18.2cal/kg/day). About 30% to about 40% should be protein, about 7% to about10% should be from carbohydrate, and about 50% to about 62.5% should bederived from fat intake. One of skill in the art can readily identifythe normal daily diet of an individual of any species.

Obesity: A condition in which excess body fat may put a person at healthrisk (see Barlow and Dietz, Pediatrics 102:E29, 1998; NationalInstitutes of Health, National Heart, Lung, and Blood Institute (NHLBI),Obes. Res. 6 (suppl. 2):51S-209S, 1998). Excess body fat is a result ofan imbalance of energy intake and energy expenditure. In one embodiment,the Body Mass Index (BMI) is used to assess obesity. In one embodiment,a BMI of 25.0 kg/m² to 29.9 kg/m² is overweight, while a BMI of 30 kg/m²is obese.

In another embodiment, waist circumference is used to assess obesity. Inthis embodiment, in men a waist circumference of 102 cm or more isconsidered obese, while in women a waist circumference of 89 cm or moreis considered obese. Strong evidence shows that obesity affects both themorbidity and mortality of individuals. For example, an obese individualis at increased risk for heart disease, non-insulin dependent (type 2)diabetes, hypertension, stroke, cancer (e.g. endometrial, breast,prostate, and colon cancer), dyslipidemia, gall bladder disease, sleepapnea, reduced fertility, and osteoarthritis, amongst others (seeLyznicki et al., Am. Fam. Phys. 63:2185, 2001).

Overweight: An individual who weighs more than their ideal body weight.An overweight individual can be obese, but is not necessarily obese. Inone embodiment, an overweight individual is any individual who desiresto decrease their weight. In another embodiment, an overweightindividual is an individual with a BMI of 25.0 kg/m² to 29.9 kg/m²

Oxyntomodulin: A further peptide produced by post-translationalprocessing of preproglucagon in the CNS and intestine, see SEQ ID NO:340. Agonists of oxyntomodulin may be identified as described above foragonists of GLP-1 and of NPY. include oxyntomodulins of other speciesand also modified sequences, for example, sequences in which The termOXM used in this text also covers any analogue of the above OXMsequence, wherein the histidine residue at position 1 is maintained orreplaced by an aromatic moiety carrying a positive charge or aderivative thereof, preferably wherein the moiety is an amino acid, morepreferably wherein it is a histidine derivative, while 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 of theother amino acids in the above OXM sequence can be independentlyreplaced by any other independently chosen amino acid, with theexception of histidine in position 1.

Any one or more (to 22) other alpha-amino acid residue in the sequencecan be independently replaced by any other one alpha-amino acid residue.Preferably, any amino acid residue other than histidine is replaced witha conservative replacement as well known in the art i.e. replacing anamino acid with one of a similar chemical type such as replacing onehydrophobic amino acid with another.

As discussed above, 1 to 22 of the amino acids can be replaced. Inaddition to the replacement option above, this may be by a non-essentialor modified or isomeric form of an amino acid. For example, 1 to 22amino acids can be replaced by an isomeric form (for example a D-aminoacid), or a modified amino acid, for example a nor-amino acid (such asnorleucine or norvaline) or a non-essential amino acid (such astaurine). Furthermore, 1 to 22 amino acids may be replaced by acorresponding or different amino acid linked via its side chain (forexample gamma-linked glutamic acid). For each of the replacementsdiscussed above, the histidine residue at position 1 is unaltered ordefined above.

In addition, 1, 2, 3, 4 or 5 of the amino acid residues can be removedfrom the OXM sequence with the exception of histidine at the 1 position(or as defined above). The deleted residues may be any 2, 3, 4 or 5contiguous residues or entirely separate residues.

The C-terminus of the OXM sequence may be modified to add further aminoacid residues or other moieties.

Pancreatic Polypeptide: A 36 amino acid peptide produced by the pancreasthat is has homology to PYY and NPY.

Peripheral Administration: Administration outside of the central nervoussystem. Peripheral administration does not include direct administrationto the brain. Peripheral administration includes, but is not limited tointravascular, intramuscular, subcutaneous, inhalation, oral, rectal,transdermal or intra-nasal administration

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The terms “polypeptide” or“protein” as used herein are intended to encompass any amino acidsequence and include modified sequences such as glycoproteins. The term“polypeptide” is specifically intended to cover naturally occurringproteins, as well as those which are recombinantly or syntheticallyproduced. The term “polypeptide fragment” refers to a portion of apolypeptide, for example such a fragment which exhibits at least oneuseful sequence in binding a receptor. The term “functional fragments ofa polypeptide” refers to all fragments of a polypeptide that retain anactivity of the polypeptide. Biologically functional peptides can alsoinclude fusion proteins, in which the peptide of interest has been fusedto another peptide that does not decrease its desired activity.

PYY: A peptide YY polypeptide obtained or derived from any species.Thus, PYY includes the human full length polypeptide (as set forth inSEQ ID NO: 1) and species variations of PYY, including e.g. murine,hamster, chicken, bovine, rat, and dog PYY (SEQ ID NOS: 5-12). In oneembodiment, PYY agonists do not include NPY. PYY also includes PYY₃₋₃₆.A “PYY agonist” is any compound which binds to a receptor thatspecifically binds PYY, and elicits an effect of PYY. In one embodiment,a PYY agonist is a compound that affects food intake, caloric intake, orappetite, and/or which binds specifically in a Y receptor assay orcompetes for binding with PYY, such as in a competitive binding assaywith labeled PYY. PYY agonists include, but are not limited to,compounds that bind to the Y2 receptor.

Substantially purified: A polypeptide which is substantially free ofother proteins, lipids, carbohydrates or other materials with which itis naturally associated. For example, the polypeptide may be at least50%, 80% or 90% free of other proteins, lipids, carbohydrates or othermaterials with which it is naturally associated.

Therapeutically effective amount: A dose sufficient to preventadvancement, or to cause regression of a disorder, or which is capableof relieving a sign or symptom of a disorder, or which is capable ofachieving a desired result. In several embodiments, a therapeuticallyeffect of PYY or an agonist thereof is an amount sufficient to inhibitor halt weight gain, or an amount sufficient to decrease appetite, or anamount sufficient to reduce caloric intake or food intake or increaseenergy expenditure.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Methods for Altering Food Intake, Appetite, Caloric Intake and EnergyExpenditure

A method is disclosed herein for reducing food intake by peripherallyadministering to a subject a therapeutically effective amount of PYY oran agonist of PYY and GLP-1 or an agonist thereof. In one embodiment,administration of PYY, or an agonist of PYY, and GLP-1 or an agonistthereof, results in a decrease in the amount, either the total weight orthe total volume of food. In other embodiment, administration of PYY, oran agonist thereof, and GLP-1 or an agonist thereof, results in adecrease of the intake of a food component, such as a decrease in theingestion of lipids, carbohydrates, cholesterol, or proteins. In the anyof the methods disclosed herein, a preferred compound, PYY₃₋₃₆ can beadministered. This disclosure includes the corresponding uses of PYY oran agonist thereof and GLP-1 or an agonist thereof, for the manufactureof a medicament or medicaments for the purposes set herein, and includesthe use of PYY₃₋₃₆.

A method is also disclosed herein for reducing caloric intake byperipherally administering to a subject a therapeutically effectiveamount of PYY or an agonist of PYY and GLP-1 or an agonist thereof. Inone embodiment, total caloric intake is reduced by peripheraladministration of a therapeutically effective amount of PYY and GLP-1 oran agonist thereof. In other embodiments, the caloric intake from theingestion of a specific food component, such as, but not limited to, theingestion of lipids, carbohydrates, cholesterol, or proteins, isreduced.

In an additional embodiment, a method is disclosed herein for reducingappetite by administering a therapeutically effective amount of PYY oran agonist thereof and GLP-1 or an agonist thereof. Appetite can bemeasured by any means known to one of skill in the art. For example,decreased appetite can be assessed by a psychological assessment. Inthis embodiment, administration of PYY and the other agent(s) results ina change in perceived hunger, satiety, and/or fullness. Hunger can beassessed by any means known to one of skill in the art. In oneembodiment, hunger is assessed using psychological assays, such as by anassessment of hunger feelings and sensory perception using aquestionnaire, such as, but not limited to, a Visual Analog Score (VAS)questionnaire (see the Examples section). In one specific, non-limitingexample, hunger is assessed by answering questions relating to desirefor food, drink, prospective food consumption, nausea, and perceptionsrelating to smell or taste.

In a further embodiment, a method is disclosed herein for alteringenergy metabolism in a subject. The method includes peripherallyadministering a therapeutically effective amount of PYY or an agonistthereof and GLP-1 or an agonist thereof, to the subject, therebyaltering energy expenditure. Energy is burned in all physiologicalprocesses. The body can alter the rate of energy expenditure directly,by modulating the efficiency of those processes, or changing the numberand nature of processes that are occurring. For example, duringdigestion the body expends energy moving food through the bowel, anddigesting food, and within cells, the efficiency of cellular metabolismcan be altered to produce more or less heat. In a further embodiment amethod is disclosed herein for any and all manipulations of the arcuatecircuitry described in this application, that alter food intakecoordinately and reciprocally alter energy expenditure. Energyexpenditure is a result of cellular metabolism, protein synthesis,metabolic rate, and calorie utilization. Thus, in this embodiment,peripheral administration of PYY and administration of GLP-1 or anagonist thereof, results in increased energy expenditure, and decreasedefficiency of calorie utilization. In one embodiment, a therapeuticallyeffective amount of PYY or an agonist and GLP-1 or an agonist thereof,thereof is administered to a subject, thereby increasing energyexpenditure.

In several embodiments, PYY (e.g., PYY₃₋₃₆) or an agonist thereof andGLP-1 or an agonist thereof, are used for weight control and treatment,reduction or prevention of obesity, in particular any one or more of thefollowing: preventing and reducing weight gain; inducing and promotingweight loss; and reducing obesity as measured by the Body Mass Index.The disclosure further relates to the use of PYY or an agonist and GLP-1or an agonist thereof, in control of any one or more of appetite,satiety and hunger, in particular any one or more of the following:reducing, suppressing and inhibiting appetite; inducing, increasing,enhancing and promoting satiety and sensations of satiety; and reducing,inhibiting and suppressing hunger and sensations of hunger. Thedisclosure further relates to the use of PYY an agonist thereof andGLP-1 or an agonist thereof, in maintaining any one or more of a desiredbody weight, a desired Body Mass Index, a desired appearance and goodhealth.

The subject can be any subject, including both human and veterinarymammalian subjects. Thus, the subject can be a human, or can be anon-human primate, a farm animal such as swine, cattle, and poultry, asport animal or pet such as dogs, cats, horses, hamsters, rodents, or azoo animal such as lions, tigers, or bears.

Obesity is currently a poorly treatable, chronic, essentiallyintractable metabolic disorder. A therapeutic drug useful in weightreduction of obese persons could have a profound beneficial effect ontheir health. Thus, the subject can be, but is not limited to, a subjectwho is overweight or obese. In one embodiment, the subject has, or is atrisk of having, a disorder wherein obesity or being overweight is a riskfactor for the disorder. Disorders of interest include, but are notlimited to, cardiovascular disease, (including, but not limited to,hypertension, atherosclerosis, congestive heart failure, anddyslipidemia), stroke, gallbladder disease, osteoarthritis, sleep apnea,reproductive disorders such as, but not limited to, polycystic ovariansyndrome, cancers (e.g., breast, prostate, colon, endometrial, kidney,and esophagus cancer), varicose veins, acnthosis nigricans, eczema,exercise intolerance, insulin resistance, hypertensionhypercholesterolemia, cholithiasis, osteoarthritis, orthopedic injury,insulin resistance (such as, but not limited to, type 2 diabetes andsyndrome X) and tromboembolic disease (see Kopelman, Nature 404:635-43;Rissanen et al., British Med. J. 301, 835, 1990).

Other associated disorders also include depression, anxiety, panicattacks, migraine headaches, PMS, chronic pain states, fibromyalgia,insomnia, impulsivity, obsessive compulsive disorder, and myoclonus.Obesity is a recognized risk factor for increased incidence ofcomplications of general anesthesia. (See e.g., Kopelman, Nature404:635-43, 2000). It reduces life span and carries a serious risk ofco-morbidities listed above.

Other diseases or disorders associated with obesity are birth defects(maternal obesity associated with increased incidence of neural tubedefects), carpal tunnel syndrome (CTS), chronic venous insufficiency(CVI), daytime sleepiness, deep vein thrombosis (DVT), end stage renaldisease (ESRD), gout, heat disorders, impaired immune response, impairedrespiratory function, infertility, liver disease, lower back pain,obstetric and gynecologic complications, pancreatititis, as well asabdominal hernias, acanthosis nigricans, endocrine abnormalities,chronic hypoxia and hypercapnia, dermatological effects, elephantitis,gastroesophageal reflux, heel spurs, lower extremity edema, mammegaly(causing considerable problems such as bra strap pain, skin damage,cervical pain, chronic odors and infections in the skin folds under thebreasts, etc.), large anterior abdominal wall masses (abdominalpanniculitis with frequent panniculitis, impeding walking, causingfrequent infections, odors, clothing difficulties, low back pain),musculoskeletal disease, pseudo tumor cerebri (or benign intracranialhypertension), and sliding hiatil hernia.

The present disclosure relates to treating, prevention, ameliorating oralleviating conditions or disorders caused by, complicated by, oraggravated by a relatively high nutrient availability. By “condition ordisorder which can be alleviated by reducing caloric (or nutrient)availability,” it is meant any condition or disorder in a subject thatis either caused by, complicated by, or aggravated by a relatively highnutrient availability, or that can be alleviated by reducing nutrientavailability, for example by decreasing food intake. Subjects who areinsulin resistant, glucose intolerant, or have any form of diabetesmellitus (e.g., type 1, 2 or gestational diabetes) can also benefit fromthis disclosure.

Such conditions or disorders are disorders associated with increasedcaloric intake, insulin resistance, or glucose intolerance and include,but are not limited to, obesity, diabetes, including type 2 diabetes,eating disorders, insulin-resistance syndromes, and Alzheimer's disease.

In another embodiment, the subject is a subject who desires weight loss,such as female and male subject who desire a change in their appearance.In yet a further embodiment, the subject is a subject who desiresdecreased feelings of hunger, such as, but not limited to, a personinvolved in a lengthy task that requires a high level of concentration(e.g., soldiers on active duty, air traffic controllers, or truckdrivers on long distance routes, etc.).

A suitable administration format may be best determined by the subjector by a medical practitioner. In one embodiment, the pharmaceuticalcompositions that include PYY, or an agonist thereof, will preferably beformulated in unit dosage form, suitable for individual administrationof precise dosages. An effective amount of PYY or an agonist thereof canbe administered in a single dose, or in multiple doses, for exampledaily, during a course of treatment. In one embodiment, PYY isadministered whenever the effect (e.g., appetite suppression, decreasedfood intake, or decreased caloric intake) is desired. In anotherembodiment, PYY or an analog thereof is administered slightly prior towhenever the effect is desired, such as, but not limited to about 10minutes, about 15 minutes, about 30 minutes, about 60 minutes, about 90minutes, or about 120 minutes, prior to the time the effect is desired.In another embodiment, a time release formulation is utilized.

In one embodiment, a therapeutically effective amount of PYY or anagonist thereof is administered as a single pulse dose, as a bolus dose,or as pulse doses administered over time. Thus, in pulse doses, a bolusadministration of PYY is provided, followed by a time period wherein noPYY is administered to the subject, followed by a second bolusadministration. In specific, non-limiting examples, pulse doses of PYYare administered during the course of a day, during the course of aweek, or during the course of a month.

The therapeutically effective amount of PYY or an agonist thereof willbe dependent on the molecule utilized, the subject being treated, theseverity and type of the affliction, and the manner of administration.For example, a therapeutically effective amount of PYY or an agonistthereof can vary from about 0.01 μg per kilogram (kg) body weight toabout 1 g per kg body weight, such as about 1 μg to about 5 mg per kgbody weight, or about 5 μg to about 1 mg per kg body weight. In anotherembodiment, PYY or an agonist thereof is administered to a subject at0.5 to 135 picomole (pmol) per kg body weight, or about 72 pmol per kgbody weight. In one specific, non-limiting example about 5 to about 50nmol is administered as a subcutaneous injection, such as about 2 toabout 20 nmol, or about 10 nmol is administered as a subcutaneousinjection. The exact dose is readily determined by one of skill in theart based on the potency of the specific compound (such as the PYYpolypeptide, or agonist) utilized, the age, weight, sex andphysiological condition of the subject. The dose of an agonist can be amolar equivalent of the therapeutically effective dose of PYY orPYY₃₋₃₆.

The compositions or pharmaceutical compositions can be administered byany route, including intravenous, intraperitoneal, subcutaneous,sublingual, transdermal, intramuscular, oral, topical, transmucosal, orby pulmonary inhalation. Compositions useful in the disclosure mayconveniently be provided in the form of formulations suitable forparenteral (including intravenous, intramuscular and subcutaneous),nasal or oral administration. The term “parenteral” as used hereinrefers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion. PYY, including PYY₃₋₃₆, anagonist of PYY, or an antagonist of PYY, can be administeredsubcutaneously. It is well known in the art that subcutaneous injectionscan be easily self-administered.

In some cases, it will be convenient to provide a PYY or a PYY agonistand another food-intake-reducing, plasma glucose-lowering or plasmalipid-altering agent, in a single composition or solution foradministration together. In other cases, it may be more advantageous toadminister the additional agent separately from said PYY or PYY agonist.

A suitable administration format may best be determined by a medicalpractitioner for each patient individually. Various pharmaceuticallyacceptable carriers and their formulation are described in standardformulation treatises, e.g., Remington's Pharmaceutical Sciences by E.W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of ParenteralScience and Technology, Technical Report No. 10, Supp. 42:2 S, 1988.

PYY and PYY agonists useful in the methods of this disclosure can beprovided as parenteral compositions, e.g., for injection or infusion.Preferably, they are suspended in an aqueous carrier, for example, in anisotonic buffer solution at a pH of about 3.0 to about 8.0, preferablyat a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0.Useful buffers include sodium citrate-citric acid and sodiumphosphate-phosphoric acid, and sodium acetate/acetic acid buffers. Aform of repository or “depot” slow release preparation may be used sothat therapeutically effective amounts of the preparation are deliveredinto the bloodstream over many hours or days following transdermalinjection or delivery.

Since the PYY and agonists are amphoteric, they may be utilized as freebases, as acid addition salts or as metal salts. The salts must, ofcourse, be pharmaceutically acceptable, and these will include metalsalts, particularly alkali and alkaline earth metal salts, e.g.,potassium or sodium salts. A wide variety of pharmaceutically acceptableacid addition salts are available. Such products are readily prepared byprocedures well known to those skilled in the art.

For use by the physician, the compositions can be provided in dosageunit form containing an amount of a PYY or a PYY agonist with or withoutanother active ingredient, e.g., a food intake-reducing, plasmaglucose-lowering or plasma lipid-altering agent. Administration maybegin whenever the suppression of nutrient availability, food intake,weight, blood glucose or plasma lipid lowering is desired, for example,at the first sign of symptoms of a weight-related disorder or shortlyafter diagnosis of obesity, diabetes mellitus, or insulin resistancesyndrome.

Therapeutically effective amounts of a PYY or a PYY agonist for use inreducing nutrient availability are those that suppress appetite at adesired level. As will be recognized by those in the field, an effectiveamount of therapeutic agent will vary with many factors including thepotency of the particular compound, age and weight of the patient, thepatient's physical condition, the blood sugar level, the weight level tobe obtained, and other factors. As will be recognized by those in thefield, an effective amount of this therapeutic agent will also vary withmany factors including the potency of the particular compound, age andweight of the patient, the patient's physical condition, the blood sugarlevel, the weight level to be obtained, and other factors.Administration may begin whenever the increased of nutrientavailability, food intake, weight, blood glucose or plasma lipidlowering is desired, such as, but not limited to, at the first sign ofsymptoms of a anorexia or at the onset of weight loss due to AIDS.

The optimal formulation and mode of administration of PYY or a PYYagonist to a patient depend on factors known in the art such as theparticular disease or disorder, the desired effect, and the type ofpatient. While the PYY and PYY agonists will typically be used to treathuman subjects they may also be used to treat similar or identicaldiseases in other vertebrates such as other primates, farm animals suchas swine, cattle and poultry, and sport animals and pets such as horses,dogs and cats.

As a pharmaceutical medicament the PYY and PYY agonists of the presentdisclosure may be administered directly by any suitable technique,including parenterally, intranasally, orally, or by absorption throughthe skin. The specific route of administration of each agent willdepend, e.g., on the medical history of the animal.

For parenteral administration, in one embodiment, PYY and PYY agonistscan be formulated generally by mixing it at the desired degree ofpurity, in a unit dosage injectable form (solution, suspension, oremulsion), with a pharmaceutically acceptable carrier, i.e., one that isnon-toxic to recipients at the dosages and concentrations employed andis compatible with other ingredients of the formulation.“Pharmaceutically acceptable carrier” refers to a non-toxic solid,semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to PYY and PYY agonists.

Generally, the formulations are prepared by contacting the PYY or PYYagonist uniformly and intimately with liquid carriers or finely dividedsolid carriers or both. Then, if necessary, the product is shaped intothe desired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

PPY and PYY agonists are also suitably administered by sustained-releasesystems. Suitable examples of sustained-release PYY and PYY agonistsinclude suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).Sustained-release PPY and PYY agonist compositions may be administeredorally, rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray.

Sustained release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556, 1983,poly(2-hydroxyethyl methacrylate)); (Langer et al., J. Biomed. Mater.Res. 15:167-277, 1981; Langer, Chem. Tech. 12:98-105, 1982, ethylenevinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid(EP 133,988).

Sustained-release PPY and PYY agonists include liposomally PPY and PYYagonists (see generally, Langer, Science 249:1527-1533, 1990; Treat etal., in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and353-365, 1989). Liposomes containing PPY peptide and peptide analogs areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. U.S.A. 82:3688-3692, 1985; Hwang et al., Proc. Natl.Acad. Sci. U.S.A. 77:4030-4034, 1980; EP 52,322; EP 36,676; EP 88,046;EP 143,949; EP 142,641; Japanese Patent Application No. 83-118008; U.S.Pat. No. 4,485,045, U.S. Pat. No. 4,544,545; and EP 102,324. Ordinarily,the liposomes are of the small (about 200-800 Angstroms) unilamellartype in which the lipid content is greater than about 30 mole percentcholesterol, the selected proportion being adjusted for the optimalperformance.

Preparations for administration can be suitably formulated to givecontrolled release of PYY and PYY agonists. For example, thepharmaceutical compositions may be in the form of particles comprising abiodegradable polymer and/or a polysaccharide jellifying and/orbioadhesive polymer, an amphiphilic polymer, an agent modifying theinterface properties of the particles and a pharmacologically activesubstance. These compositions exhibit certain biocompatibility featureswhich allow a controlled release of the active substance. See U.S. Pat.No. 5,700,486.

In yet an additional embodiment, PPY and PYY agonists are delivered byway of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng.14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudek et al., N.Engl. J. Med. 321:574, 1989) or by continuous subcutaneous infusions,for example, using a mini-pump. An intravenous bag solution may also beemployed. The key factor in selecting an appropriate dose is the resultobtained, as measured by decreases in total body weight or ratio of fatto lean mass, or by other criteria for measuring control or preventionof obesity or prevention of obesity-related conditions, as are deemedappropriate by the practitioner. Other controlled release systems arediscussed in the review by Langer (Science 249:1527-1533, 1990).

In another aspect of the disclosure, PPY and PYY agonists are deliveredby way of an implanted pump, described, for example, in U.S. Pat. No.6,436,091; U.S. Pat. No. 5,939,380; U.S. Pat. No. 5,993,414.

Implantable drug infusion devices are used to provide patients with aconstant and long term dosage or infusion of a drug or any othertherapeutic agent. Essentially such device may be categorized as eitheractive or passive.

Active drug or programmable infusion devices feature a pump or ametering system to deliver the drug into the patient's system. Anexample of such an active drug infusion device currently available isthe Medtronic SynchroMed™ programmable pump. Such pumps typicallyinclude a drug reservoir, a peristaltic pump to pump out the drug fromthe reservoir, and a catheter port to transport the pumped out drug fromthe reservoir via the pump to a patient's anatomy. Such devices alsotypically include a battery to power the pump as well as an electronicmodule to control the flow rate of the pump. The Medtronic SynchroMed™pump further includes an antenna to permit the remote programming of thepump. Passive drug infusion devices, in contrast, do not feature a pump,but rather rely upon a pressurized drug reservoir to deliver the drug.Thus such devices tend to be both smaller as well as cheaper as comparedto active devices. An example of such a device includes the MedtronicIsoMed™. This device delivers the drug into the patient through theforce provided by a pressurized reservoir applied across a flow controlunit.

The implanted pump can be completely implanted under the skin of apatient, thereby negating the need for a percutaneous catheter. Theseimplanted pumps can provide the patient with PYY or a PYY agonist at aconstant or a programmed delivery rate, e.g., to give pulsed doses at oraround meal time. Constant rate or programmable rate pumps are based oneither phase-change or peristaltic technology. When a constant,unchanging delivery rate is required, a constant-rate pump is wellsuited for long-term implanted drug delivery. If changes to the infusionrate are expected, a programmable pump may be used in place of theconstant rate pump system. Osmotic pumps may be much smaller than otherconstant rate or programmable pumps, because their infusion rate can bevery low. An example of such a pump is described listed in U.S. Pat. No.5,728,396.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

For administration by inhalation, the compounds for use according to thepresent disclosure are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Pharmaceutical compositions that comprise a PYY, or an agonist thereof,as described herein as an active ingredient will normally be formulatedwith an appropriate solid or liquid carrier, depending upon theparticular mode of administration chosen. The pharmaceuticallyacceptable carriers and excipients useful in this disclosure areconventional. For instance, parenteral formulations usually compriseinjectable fluids that are pharmaceutically and physiologicallyacceptable fluid vehicles such as water, physiological saline, otherbalanced salt solutions, aqueous dextrose, glycerol or the like.Excipients that can be included are, for instance, other proteins, suchas human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered may also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate. Other medicinal andpharmaceutical agents, for instance other appetite suppressants, orprotease inhibitors, also may be included. Actual methods of preparingsuch dosage forms are known, or will be apparent, to those skilled inthe art.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, inhalation, suppository, and oral formulations can beemployed. The pharmaceutical compositions can be produced ofconventional mixing, granulating, confectioning, dissolving orlyophilizing processes.

Oral formulations may be liquid (e.g., syrups, solutions orsuspensions), or solid (e.g., powders, pills, tablets, or capsules). Forexample, pharmaceutical compositions for oral use can be obtained bycombining the active ingredient with one or more solid carriers,optionally granulating a resulting mixture, and, if desired, processingthe mixture or granules, if appropriate with the addition of additionalexcipients, to form tablets or dragee cores.

Suitable carriers include fillers, such as sugars, for example lactose,saccharose, mannitol or sorbitol, cellulose preparations and/or calciumphosphates, for example tricalcium phosphate or calcium hydrogenphosphate, also binders, such as starches, for example corn, wheat, riceor potato starch, methylcellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose and/or polyvinylpyffolidone, and/or, if desired,disintegrators, such as the above-mentioned starches, also carboxymethylstarch, cross-linked polyvinylpyrrolidone, alginic acid or a saltthereof, such as sodium alginate. Additional excipients include flowconditioners and lubricants, for example silicic acid, talc, stearicacid or salts thereof, such as magnesium or calcium stearate, and/orpolyethylene glycol, or derivatives thereof.

For parenteral administration compositions include suitable aqueoussolutions of an active ingredient in water-soluble form, for example inthe form of a water-soluble salt, or aqueous injection suspensions thatcontain viscosity-altering substances, for example sodiumcarboxymethylcellulose, sorbitol and/or dextran, and, if desired,stabilizers. The active ingredient, optionally together with excipients,can also be in the form of a lyophilisate and can be made into asolution prior to parenteral administration by the addition of suitablesolvents. Solutions such as those that are used, for example, forparenteral administration can also be used as infusion solutions.

For inhalation, PYY or an agonist thereof is administered as an aerosolor a dispersion in a carrier. In one specific, non-limiting example, PYYor an agonist thereof is administered as an aerosol from a conventionalvalve, such as, but not limited to, a metered dose valve, through anaerosol adapter also known as an actuator. A suitable fluid carrier canbe also included in the formulation, such as, but not limited to, air, ahydrocarbon, such as n-butane, propane, isopentane, amongst others, or apropellant, such as, but not limited to a fluorocarbon. Optionally, astabilizer is also included, and/or porous particles for deep lungdelivery are included (e.g., see U.S. Pat. No. 6,447,743).

Compounds with poor solubility in aqueous systems require formulation byusing solubilizing agents such as ionic surfactants, cholates,polyethylene glycol (PEG), ethanol, or other agents which may haveundesirable effects when used for inhalation. In addition, a treatmentrequiring successful delivery into alveoli of the lower pulmonary regionmay preclude from the formulation the use of certain irritants such aschlorofluorocarbons and should involve a minimum number of requireddoses. Alternatively, to avoid such limitations, liposomes orhydrophobic particles can be used. In one embodiment, an inhalationformulation for a sustained release includes using aerosol dropletparticles approximately 1-2.1 μm in size, or of less than 1 μm in size.Small particle aerosol liposomes and liposome-drug combinations formedical use have been previously described (e.g., see EP 87309854.5).

According to the invention, a therapeutically effective amount of PYY oran agonist thereof is administered with a therapeutically effectiveamount of GLP-1 or an agonist thereof.

The PYY or agonist thereof and the GLP-1 or an agonist thereof may beadministered simultaneously or substantially simultaneously, orsequentially, in any order. The PYY or agonist thereof and the GLP-1 oran agonist thereof may be administered in a single pharmaceuticalcomposition or in separate compositions, and they may be administered bythe same route or my different routes.

If the PYY and the GLP-1 or an agonist thereof are to be administered ina single pharmaceutical composition, that composition may be any ofthose described above for PYY or an agonist thereof. The composition mayenable simultaneous or substantially simultaneous administration of thePYY or agonist thereof and the GLP-1 or an agonist thereof. If desired,the PYY or agonist thereof and the GLP-1 or an agonist thereof may becompartmentalized in the composition, for example, in different layersof a tablet, or in different granules in a capsule. If desired, suchcompartmentalization may be designed to give different releaseproperties to the two components to enable delivery of the PYY oragonist component and the GLP-1 or an agonist thereof at differenttimes, for example, sequentially.

Alternatively, the PYY or agonist thereof and the GLP-1 or an agonistthereof may be formulated in separate pharmaceutical compositions, forexample, any of the pharmaceutical compositions described above for PYYand agonists thereof. Such separate compositions may be administeredsimultaneously or substantially simultaneously, or they may beadministered sequentially, in any order.

If administered separately, whether sequentially or simultaneously (orsubstantially simultaneously), the PYY or agonist thereof and the GLP-1or an agonist thereof may be administered by the same route or bydifferent routes. It is generally more convenient to administer all theactive agents in a single composition. However, in some cases it may benecessary or appropriate to administer the active agents by differentroutes. For example, peptides are generally not stable on oraladministration unless modified or formulated in a special way, so mustgenerally be administered via a non-oral route. Some agonists, forexample, GLP-1 agonists, are chemical compounds that are stable whenadministered orally. It may be appropriate to administer OXM non-orallyand the other component by a non-oral route.

When PYY or an agonist thereof and GLP-1 or an agonist thereof are usedin the manufacture of a medicament for use in a treatment as describedherein, the medicament may be a single pharmaceutical compositioncomprising both components, as described above, or may be atwo-component medicament, one component being a pharmaceuticalcomposition comprising PYY or an agonist thereof, the other componentbeing a pharmaceutical composition comprising GLP-1 or an agonistthereof, see above. The medicament, whether a one component medicamentor a two component medicament as described above, will generally bepackaged with instructions relating to its use. Such instructions willrefer to the timing, dose and route of administration of thecomponent(s).

If desired, a further agent that influences food intake and/or appetitemay also be administered. Such agents include naturally-occurring agentsand agonists thereof, for example, amylin and amylin agonists, leptinand leptin agonosts, and oxyntomodulin.

If desired, one or more other agents, such as, but not limited to, anadditional appetite suppressant, may also be administered. Specific,non-limiting example of an additional appetite suppressant includeamfepramone (diethylpropion), phentermine, mazindol andphenylpropanolamine, fenfluramine, dexfenfluramine, and fluoxetine.

PYY or a PYY agonist, or GLP-1 or a GLP-1 agonist, or both, can beadministered simultaneously with the additional agent(s), or the varioussubstances may be administered sequentially, and in any order. Thus, inone embodiment, PYY or an agonist thereof and GLP-1 or an agonistthereof is formulated and administered with an additional agent, forexample, oxyntomodulin or an agonist thereof, or an appetite suppressantas a single composition.

Additionally, a method of treating obesity is disclosed herein. Themethod includes administering to an obese subject a therapeuticallyeffective amount of PYY or a PYY agonist, and GLP-1 or an agonistthereof. The PYY agonist can have potency in at least one of food intakeor gastric emptying greater than NPY. PYY or a PYY agonist can beadministered peripherally, such as in a single or divided dose. Suitablesingle or divided doses include, but are not limited to, 1 μg to about 5mg or about 0.01 μg/kg to about 500 μg/kg per dose. The subject can beinsulin resistant or glucose intolerant, or both. In addition to beingobese, the subject can have diabetes mellitus.

A method of reducing food intake is also disclosed herein. The methodincludes administering to an obese subject a therapeutically effectiveamount of PYY or a PYY agonist, and GLP-1 or an agonist thereof. The PYYagonist can have potency in at least one of food intake or gastricemptying greater than NPY. PYY or a PYY agonist can be administeredperipherally, such as in a single or divided dose. Suitable single ordivided doses include, but are not limited to, 1 μg to about 5 μg orabout 0.01 μg/kg to about 500 μg/kg per dose. The subject can have TypeII diabetes, and/or can be overweight.

A method is disclosed herein for improving lipid profile in a subject.The method includes administering to the subject an effective amount ofPYY or a PYY agonist, and GLP-1 or an agonist thereof. An improvement inlipid profile includes, but is not limited to, at least one method ofreducing cholesterol levels, reducing triglyceride levels and increasingHDL cholesterol levels. PYY or a PYY agonist can be administeredperipherally, such as in a single or divided dose. PYY or a PYY agonistcan be administered peripherally, such as in a single or divided dose.Suitable single or divided doses include, but are not limited to, 1 μgto about 5 mg or about 0.01 μg/kg to about 500 μg/kg per dose. The PYYagonist can have potency in at least one of food intake or gastricemptying greater than NPY.

In another embodiment, a method is disclosed herein for alleviating acondition or disorder which can be alleviated by reducing nutrientavailability. The method includes administering to a subject atherapeutically effective amount of PYY or a PYY agonist, and GLP-1 oran agonist thereof. Suitable disorders include any of the disordersmentioned above.

The PYY or a PYY agonist and GLP-1 or GLP-1 agonist can be administeredperipherally, such as in a single or divided dose. Suitable single ordivided doses include, but are not limited to, 1 μg to about 5 mg orabout 0.01 μg/kg to about 500 μg/kg of PYY or a PYY agonist per dose.The PYY agonist can have potency in at least one of food intake orgastric emptying greater than NPY. Suitable doses also include thosethat raise the concentration of PYY and/or the agonist thereofsignificantly above the basal concentration of PYY, such as, but notlimited to, a dose that that mimic postparandial serum concentrations ofPYY (or the agonist). Thus, in one embodiment, PYY or an agonist thereofis administered to achieve the level of to effect a reduction in calorieintake, food intake, or appetite equivalent to the reduction in calorieintake, food intake, or appetite, or to increase the energy expenditure,caused by the postprandial level of PYY₃₋₃₆. Specific, non-limitingexamples of doses include, but are not limited doses that produce theeffect demonstrated when the serum levels of PYY are from about 40pM/litre to about 60 pM/litre, for example, from about 40 pM/litre toabout 50 pM/litre, or from about 40 pM/litre to about 45 pM/litre, or toabout 43 pM/litre.

For all methods disclosed herein, the dose of PYY or PYY₃₋₃₆ can bebased on the physiological levels observed post-prandially. The normalcirculating levels of PYY₃₋₃₆ are about 8 pmol/litre, typically risingto about 40 to 60 pmol/litre after a meal. Agonists of PYY can be usedat analogous doses. A single dose may be administered per day, ordivided doses can be used (see above).

As indicated above, PYY₃₋₃₆ has a prolonged and sustained action,continuing to act even after it has been cleared from the circulatingblood, for example, for up to 12 and even up to 24 hours. Accordingly,PYY or an agonist thereof may be administered twice per day or even justonce. If desired, more doses may be administered, for example, three orfour per day. For example, it may be desired to administer PYY or anagonist thereof before a meal, for example, about half an hour before ameal. It may be desired to administer PYY or an agonist thereof beforeeach meal. Alternately, it may be desired to administer as few doses aspossible, in which case a regime or one or two doses per day may beused.

GLP-1 or an agonist may be administered according to the same regime asthe PYY or agonist thereof, whether administered simultaneously orsubstantially simultaneously with the PYY or PYY agonist, orsequentially. Alternatively, the GLP-1 may be administered in adifferent regime, for example, with more doses of GLP-1 than of PYY, forexample, the PYY or PYY agonist may be administered once or twice perday, and the GLP-1 or agonist thereof may be administered three or fourtimes per day. For example, PYY or a PYY agonist may be administered inthe morning and evening, for example, before breakfast and before theevening meal, and the GLP-1 agonist thereof may also be administeredbefore the mid-day meal.

In one embodiment, when administered peripherally, PYY, includingPYY₃₋₃₆ has its effects at physiological levels. No side effects areobserved when PYY₃₋₃₆ is used at such levels. Without being bound bytheory, PYY₃₋₃₆ does not affect Y2 receptors throughout the brain, whichcould cause side effects. It should be noted, without being limiting,that a further advantage of PYY₃₋₃₆ is that PYY₃₋₃₆ does not increaseblood pressure. The effects of PYY₃₋₃₆ are as long lasting as 24 hours.Recipients claim a decrease in appetite over that period, and areduction of food intake of about one third has been reported.

In one specific, non-limiting example, PYY₃₋₃₆ is administered in a doseof about 1 nmol or more, 2 nmol or more, or 5 nmol or more. In thisexample, the dose of PYY₃₋₃₆ is generally not more than 100 nmol, forexample, the dose is 90 nmols or less, 80 nmols or less, 70 nmols orless, 60 nmols or less, 50 nmols or less, 40 nmols or less, 30 nmols orless, 20 nmols or less, 10 nmols. For example, a dosage range maycomprise any combination of any of the specified lower dose limits withany of the specified upper dose limits. Thus, exemplar non-limiting doseranges include a dose of PYY₃₋₃₆ may be within the range of form 1 to100 n mols, from 1 to 90 mols, from 1 to 80 nmols. Exemplary,non-limiting dose ranges include, from 2 to 100 nmols, from 2 to 90 nmols, for example, from 2 to 80 nmols etc., from 5 nmols to 100 mols,from 5 nmols to 90 nmols, from 5 nmols to 80 nmols etc. By way ofexample, a dose of from about 5 to about 50 nmol may be administeredsuch as, but not limited to, from about 2 to about 20 nmol, for example,about 10 nmol. The selected dose may be administered for example, byinjection, for example, as a subcutaneous injection. A PYY agonist maybe administered at the same dose.

In a further, non-limiting example, PYY₃₋₃₆ may be administeredperipherally at a dose of 0.1 nmoles or more per kg body weight of thesubject, for example, 0.2 nmoles or more, for example, 0.4 nmoles ormore, for example, 0.6 nmoles or more, for example, 0.8 nmoles or more,for example, 1.0 nmole or more, for example, 1.2 nmoles or more, forexample, 1.4 nmoles or more, for example, 1.6 nmoles or more, forexample, 1.8 nmoles or more, for example, 2.0 nmoles or more, forexample, 2.2 nmoles or more, for example, 2.4 nmoles or more, forexample, 2.6 nmoles or more, for example, 2.8 nmoles, for example, 3.0nmoles or more, for example, up to 3.2 nmoles per kg body weight.PYY₃₋₃₆ or a PYY agonist and may be administered peripherally in anamount of up to 3.0 nmoles per kg body weight, for example, up to 2.8nmoles, for example, up to 2.6 nmoles, for example, up to 2.4 nmoles,for example, up to 2.2 nmoles, for example, up to 2.0 nmoles, forexample, up to 1.8 nmoles, for example, up to 1.4 nmoles, for example,up to 1.2 nmoles, for example, up to 1.0 nmoles, for example, up to 0.8nmoles, for example, up to 0.6 nmoles, for example, up to 0.4 nmoles,for example, up to 0.2 nmoles per kg body weight.

For subcutaneous administration, a dose of PYY for example PYY₃₋₃₆ or aPYY agonist of 10 nmoles or more, for example, 20 nmoles or more, forexample, 30 nmoles or more, for example, 40 nmoles or more may be used.It is generally preferable to use up to about 70 nmoles, for example, upto about 60 mmoles. A dose of about 40 nmoles may be used. In oneembodiment, a dose of PYY or PYY₃₋₃₆ at 0.143 n moles ( 1/7^(th) of amole) is administered per kilogram, to achieve a dose that is similar tothe postprandial level of PYY.

If PYY itself or an agonist thereof is used, the dose is preferably amolar equivalent of a PYY₃₋₃₆ dose, or achieves the same effect as aPYY₃-36 dose, as described above. The doses can be calculated on thebasis of a subject, such as a subject weighing from 70 to 75 kg. Theexact dose is readily determined by one of skill in the art based on thepotency of the specific compound (such as the PYY polypeptide, oragonist) utilized, and the age, weight, sex and physiological conditionof the subject.

All the teachings given above in relation to PYY₃₋₃₆ also apply to PYYitself and to PYY agonists. For example, the teachings relating toroutes of administration and dosage regimes.

The GLP-1 or an agonist thereof should be administered in antherapeutically effective dose, see the section relating to GLP-1 andagonists thereof below. GLP-1 or an agonist thereof may be used inamounts or doses in the ranges described above for PYY or a PYY agonist.The doses of the various agent may be independent of each other or, forexample, equimolar doses may be used, for example, equimolar doses ofGLP-1 or an agonist thereof and PYY or an agonist thereof.

As disclosed herein, a naturally occurring peptide, PYY or PYY₃₋₃₆ canbe used to achieve a physiological effect. This results in minimal sideeffects and enables long term use, if necessary. The dose of PYY orPYY₃₋₃₆ can be based on the physiological levels observedpost-prandially. The normal circulating levels of PYY₃₋₃₆ are about 8pmol/litre, typically rising to about 40 to 60 pmol/litre after a meal.PYY (e.g., PYY₃₋₃₆) and agonists can be used at analogous doses.

The considerations set out above in relation to the administrationregime also relate to administration using the subcutaneous route.

The various uses of PYY, or an agonist thereof, and GLP-1 or an agonistthereof, as set out above may be in a method of treatment of a mammaliansubject in need of such treatment, or may be in the manufacture of amedicament for such treatment. PYY (e.g., PYY₃₋₃₆) or an agonistthereof, and GLP-1 or an agonist thereof, should be administered in anamount effective to achieve the stated object. Some of the treatmentsdescribed above are medical treatments, for example, the treatment ofobesity. Others, however, do not relate to medical treatment, and arepart of the maintenance of a healthy lifestyle, or are for cosmeticpurposes.

The use of a combination of any of OXM and GLP-1 or an agonist thereofand PYY or an agonist thereof may serve to increase the effectiveness ofany of the agents compared with its use alone, for example, as describedabove. Alternatively or in addition, use of the two or three agents incombination may reduce any tendency for “escape” when using an agentalone. The term “escape” is used to denote a reduction in effect of anagent with time. For example, if any one of the agents above has beenused alone, its effect may reduce with time. Use of one or both of theother agents in addition may reduce or prevent the tendency for thatreduction in effectiveness. For example, PYY has a sustained effect andmay be used for prolonged periods. If the effect of PYY should appear toreduce, or to reduce or prevent any such reduction in effect, OXM may beadministered in addition to the PYY. GLP-1 may also be used for the samepurpose, with OXM or with OXM and PYY.

In another aspect the present invention provides a pharmaceuticalcomposition that comprises PYY or a PYY agonist as the active ingredientand a pharmaceutically suitable carrier in a form suitable forsubcutaneous administration, for example, as an injectable solution.Such a composition may comprise an amount of PYY for example PYY₃₋₃₆ ora PYY agonist of 10 nmoles or more, for example, 20 nmoles or more, forexample, 30 nmoles or more, for example, 40 nmoles or more may be used.It is generally preferable to use up to about 70 nmoles, for example, upto about 60 mmoles. An amount of about 40 nmoles may present. Suitableformulations for subcutaneous administration are well known, and aredescribed above. One or more further active ingredient(s) may also bepresent in the composition, in particular GLP-1 or an agonist thereof,which may be used in amounts and doses as described above. Furtheractive ingredients may be present, for example, as described above, forexample, oxyntomodulin or an agonist thereof, or an appetite suppressantas described above.

A further aspect of the invention relates to methods as disclosed above,including a method for decreasing calorie intake in a subject, a methodfor decreasing appetite in a subject, a method for decreasing foodintake in a subject, a method for weight control or treatment in asubject, and a method for reduction or prevention of obesity, inparticular any one or more of the following: preventing and reducingweight gain; inducing and promoting weight loss; and reducing obesity asmeasured by the Body Mass Index. The methods include control of any oneor more of appetite, satiety and hunger, in particular any one or moreof the following: reducing, suppressing and inhibiting appetite;inducing, increasing, enhancing and promoting satiety and sensations ofsatiety; and reducing, inhibiting and suppressing hunger and sensationsof hunger. The methods further include maintaining any one or more of adesired body weight, a desired Body Mass Index, a desired appearance andgood health. In the methods of this aspect of the invention, PYY, forexample, PYY₃₋₃₆ or a PYY agonist is administered subcutaneously to thesubject.

Preferably the PYY or PYY agonist is administered at dose of 10 nmolesor more, for example, 20 nmoles or more, for example, 30 nmoles or more,for example, 40 nmoles or more may be used. It is generally preferableto use up to about 70 nmoles, for example, up to about 60 mmoles. Andose of about 40 nmoles be used. As indicated above, PYY₃₋₃₆ has aprolonged and sustained action, continuing to act even after it has beencleared from the circulating blood, for example, for up to 12 and evenup to 24 hours. Accordingly, PYY or an agonist thereof may beadministered twice per day or even just once. If desired, more doses maybe administered, for example, three or four per day. For example, it maybe desired to administer PYY or an agonist thereof before a meal, forexample, about half an hour before a meal. It may be desired toadminister PYY or an agonist thereof before each meal. Alternately, itmay be desired to administer as few doses as possible, in which case aregime or one or two doses per day may be used.

In this aspect of the invention, PYY or an agonist thereof may beadministered alone or PYY or an agonist thereof and GLP-1 or an agonistthereof may be administered. If GLP-1 or an agonist thereof is used inaddition to the PYY or PYY agonist, the GLP-1 or agonist thereof may beadministered simultaneously or substantially simultaneously with the PYYor agonist thereof, or sequentially, in either order. The PYY or agonistthereof and the GLP-1 or agonist thereof may be administered in a singlepharmaceutical composition or in separate compositions, and they may beadministered by the same route or by different routes. Further activeingredients may also be administered, for example, as described abovefor subcutaneous PYY pharmaceutical compositions.

The present invention also includes PYY or an agonist thereof for use inthe manufacture of a medicament for subcutaneous administration for anyof the methods of treatment described above.

PYY Agonists

A PYY agonist, of use in the methods of the present disclosure, is amolecule that binds to a receptor that specifically binds PYY, andelicits an effect of PYY. Assays for binding to PYY receptors, andeliciting a response in a cell with a PYY receptor, are known in theart. A specific assay for detecting a PYY agonist is also disclosedherein. Thus, in one embodiment, a PYY agonist binds to a NPY neuron inthe arcuate nucleus, which results in an electrophysiological effect onan NPY neuron. As disclosed herein, NPY neurons synapse with POMCneurons. Thus, the electrophysiological effect on the NYP neuron canresult in a further electrophysiological effect on a POMC neuron. In onespecific, non-limiting example, an administration of PYY agonist resultsin hyperpolization of the membrane potential of a POMC neuron. Inanother specific, non-limiting example, administration of a PYY agonistresults in an increase in IPSCs in a POMC neuron.

In another embodiment, PYY agonists do not include NPY. Suitable PYYagonists include molecules that bind NPY neurons, but do not cross theblood/brain barrier. The arcuate nucleus neurons upon which PYY exertsits effects are not protected by the blood/brain barrier, and thus arereadily accessible to peripherally available molecules. In addition,other brain sites that express the Y2 receptor are protected by theblood/brain barrier. Without being bound by theory, agents able to bindto the arcuate Y2R, but that do not cross the blood/brain batherfollowing peripheral administration, are likely to be of use.

In one embodiment, a PYY agonist is a compound that affects food intake,caloric intake, or appetite, and/or which binds specifically in a Yreceptor assay or competes for binding with PYY, such as in acompetitive binding assay with labeled PYY. PYY agonists include, butare not limited to, compounds that bind to the Y2 receptor.

PYY and agonists useful in the methods disclosed herein include, but arenot limited to, polypeptides comprising, or alternatively consisting of,the amino acid sequence for PPY and agonists thereof, e.g., mutants,fragments and/or variants thereof. Variants include deletions,insertions, inversions, repeats and substitutions (e.g., conservativesubstitutions and non-conservative substitutions; see, e.g., Tables 1and 2, infra). More than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9,10, etc.) can be deleted or inserted or substituted with another aminoacid. Typically conservative substitutions are the replacements, one foranother, among the aliphatic amino acids Ala, Val, Leu and Ile;interchange of Ser and Thr containing hydroxy residues, interchange ofthe acidic residues Asp and Glu, interchange between the amide residuesAsn and Gln, interchange of the basic residues Lys and Arg, interchangeof the aromatic residues Phe and Tyr, and interchange of the small-sizedamino acids Ala, Ser, Thr, Met and Gly. Guidance concerning how to makephenotypically silent amino acid substitutions is provided in Bowie etal., Science 247:1306-1310, 1990.

As another example, polypeptide fragments may contain a continuousseries of deleted residues from the amino (N)- or the carboxyl(C)-terminus, or both (see, e.g., Tables 1 and 2, infra). Any number ofamino acids, ranging from 1 to 24, can be deleted from the N-terminus,the C-terminus or both.

Furthermore, the agonist polypeptides may also include, but are notlimited to, polypeptides comprising, or alternatively consisting of,internal deletions of the amino acid sequences for PPY and/or agonistthereof (see, e.g., Table 2, infra). Such deletions may comprise one ormore amino acid residue deletions (e.g., one, two, three, four, five,six, seven, eight, nine, ten, etc.) and may begin at any amino acidposition (e.g., two, three, four, five, six, seven, eight, nine, ten,etc.). In addition, the polypeptides of this disclosure may contain oneor more such internal deletions. Such deletions are contemplated in PPY,NPY and PP.

Also contemplated are agonist peptides that are PPY, NPY and/or PPchimeras having high affinity and/or selectivity for the Y2 receptor.These chimeras may comprise amino acid substitutions of one or moreamino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) from PPY, NPYand/or PP, variants, mutants and/or deletions thereof, with one or moreamino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) from a secondPPY, NPY, or PP, variants, mutations and/or deletions thereof. Thesesubstitutions may begin at any amino acid position (e.g., two, three,four, five, six, seven, eight, nine, ten, etc.).

Preferably, the peptide is selective for the Y2 receptor. That is, itbinds with higher affinity to Y2 compared to other receptors, such asY1, Y2, Y3, Y4, Y5 and Y6. In another embodiment, the peptide isselective for the Y2 and Y5 receptors over the Y1, Y3, Y4 and Y6receptors.

Other polypeptide fragments are fragments comprising structural orfunctional domain of the polypeptides of this disclosure. Such fragmentsinclude amino acid residues that comprise a polyproline-type II helix(residues 1-8), beta-turn (residues 9-14), amphipathic alpha-helix(residues 15-32) and/or a C-terminal turn structure (residues 33-36).See, Kirby et al., J Med Chem 36:385-393, 1993.

In addition, this disclosure includes the use of a polypeptide oragonist comprising, or alternatively consisting of, the amino acidsequence for PPY, NPY and PP species variants (see Table 1, infra)and/or mutants, and fragments thereof.

Also contemplated are fusion proteins, whereby a PYY or PYY agonist willbe fused to another protein or polypeptide (the fusion partner) usingrecombinant methods known in the art. Alternatively, such a fusionprotein may be synthetically synthesized by any known method. Any knownpeptide or protein can be used as the fusion partner (e.g., serumalbumin, carbonic anhydrase, glutathione-S-transferase or thioredoxin,etc.). Preferred fusion partners will not have an adverse biologicalactivity in vivo. Such fusion proteins may be designed linking thecarboxy-terminus of the fusion partner to the amino-terminus of the PYYor agonist peptide, or vice versa. Optionally, a cleavable linker regionmay be used linking the PYY or PYY agonist to the fusion partner, andmay be cleaved in vivo thereby resulting in the release of an activeform of PYY or a PYY agonist. Examples of such cleavage regions include,but are not limited to, the linker regions D-D-D-D-Y (SEQ ID NO: 330),G-P-R (SEQ ID NO: 331), A-G-G (SEQ ID NO: 332) and H-P-F-H-L (SEQ ID NO333), which can be cleaved by enterokinase, thrombin, ubiquitin cleavingenzyme and renin, respectfully. See, e.g., U.S. Pat. No. 6,410,707.

Also contemplated as useful PYY agonists are Y2 specific NPY peptideagonists as described in U.S. Pat. No. 5,026,685; U.S. Pat. No.5,574,010; U.S. Pat. No. 5,604,203; U.S. Pat. No. 5,696,093; U.S. Pat.No. 6,046,167. See below:

Preferred PPY agonists are described herein as follows.

TABLE 1 PYY: Variation Among Species AA SEQUENCE PEPTIDE  YY HumanYPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 1) RatYPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ ID NO: 5) PigYPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ ID NO: 6) Guinea pigYPSKPEAPGSDASPEELARYYASLRHYLNLVTRQRY (SEQ ID NO: 7) FrogYPPKPENPGEDASPEEMTKYLTALRHYINLVTRQRY (SEQ ID NO: 8) RajaYPPKPENPGDDAAPEELAKYYSALRHYINLITRQRY (SEQ ID NO: 9) DogfishYPPKPENPGEDAPPEELAKYYSALRHYINLITRQRY (SEQ ID NO: 10) LampetraFPPKPDNPGDNASPEQMARYKAAVRHYINLITRQRY (SEQ ID NO: 11) PetromyzonMPPKPDNPSPDASPEELSKYMLAVRNYINLITRQRY (SEQ ID NO: 12) NEUROPEPTIDE  YHuman YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY (SEQ ID NO: 2) RatYPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY (SEQ ID NO: 13) RabbitYPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY (SEQ ID NO: 14) DogYPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY (SEQ ID NO: 15) PigYPSKPDNPGEDAPAEDLARYYSALRHYINLITRQRY (SEQ ID NO: 16) CowYPSKPDNPGEDAPAEDLARYYSALRHYINLITRQRY (SEQ ID NO: 17) SheepYPSKPDNPGDDAPAEDLARYYSALRHYINLITRQRY (SEQ ID NO: 18) Guinea pigYPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY (SEQ ID NO: 19) AvianYPSKPDSPGEDAPAEDMARYYSALRHYINLITRQRY (SEQ ID NO: 20) RanaYPSKPDNPGEDAPAEDMAKYYSALRHYINLITRQRY (SEQ ID NO: 21) GoldfishYPTKPDNPGEGAPAEELAKYYSALRHYINLITRQRY (SEQ ID NO: 22) DogfishYPSKPDNPGEGAPAEDLAKYYSALRHYINLITRQRY (SEQ ID NO: 23) LampetraPPNKPDSPGEDAPAEDLARYLSAVRHYINLITRQRY (SEQ ID NO: 24) PANCREATICPOLYPEPTIDE Human ASLEPEYPGDNATPEQMAQYAAELRRYINMLTRPRY (SEQ ID NO: 3)Sheep APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY (SEQ ID NO: 25) PigAPLEPVYPGDDATPEQMAQYAAELRRYINMLTRPRY (SEQ ID NO: 26) DogAPLEPVYPGDDATPEQMAQYAAELRRYINMLTRPRY (SEQ ID NO: 27) CatAPLEPVYPGDNATPEQMAQYAAELRRYINMLTRPRY (SEQ ID NO: 28) CowAPLEPEYPGDNATPEQMAQYAAELRRYINMLTRPRY (SEQ ID NO: 29) RatAPLEPMYPGDYATHEQRAQYETQLRRYINTLTRPRY (SEQ ID NO: 30) MouseAPLEPMYPGDYATPEQMAQYETQLRRYINTLTRPRY (SEQ ID NO: 31) Guinea pigAPLEPVYPGDNATPEQQMAQYAAEMRRYINMLTRPRY (SEQ ID NO: 32) ChickenGPSQPTYPGDDAPVEDLIRFYNDLQQYLNVVTRHRY (SEQ ID NO: 33) AlligatorTPLQPKYPGDGAPVEDLIQFYNDLQQYLNVVTRPRF (SEQ ID NO: 34) BullfrogAPSEPHHPGDQATPDQLAQYYSDLYQYITFITRPRF (SEQ ID NO: 35) Ref:Beck-Sickinger, A. G., Jung, G., Biopolymers 37: 123-142, 1995.

TABLE 2 PEPTIDE AGONIST OF PYY PEPTIDE SEQUENCE PPY(3-36)(human)IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 334)Ref: Eberlein et al., Peptides 10: 797-803, 1989; Grandt et al.,Peptides 15(5): 815-20, 1994. Variations of PPY(3-36)N-Terminal Deletions of PYY, including but not limited to: PYY(26-36),PYY(25-36), PYY(24-36), PYY(23-36), PYY(22-36), PYY(21-36), PYY(20-36),PYY(19-36), PYY(18-36), PYY(17-36), PYY(16-36), PYY(15-36), PYY(14-36),PYY(13-36), PYY(12-36), PYY(11-36), PYY(10-36), PYY(9-36), PYY(8-36),PYY(7-36), PYY(6-36), PYY(5-36), PYY(4-36), PYY(3-36).Ref: See, e.g., Balasubramaniam et al., Pept Res 1(1): 32-5, September-October 1998; Liu et al., J Gastrointest Surg 5(2): 147-52,March-April 2001. NPY (human) YPSKPDNPGEDAPAEDMARYYSALRHYINLITRQRY(SEQ ID NO: 2)Ref: Tatemoto et al., Proc Natl Acad Sci U.S.A. 79: 5485-9, 1982.Variations of NPYN-Terminal Deletions of NPY, including but not limited to: NPY(26-36),NPY(25-36), NPY(24-36), NPY(23-36), NPY(22-36), NPY(21-36), NPY(20-36),NPY(19-36), NPY(18-36), NPY(17-36), NPY(16-36), NPY(15-36), NPY(14-36),NPY(13-36), NPY(12-36), NPY(11-36), NPY(10-36), NPY(9-36), NPY(8-36),NPY(7-36), NPY(6-36), NPY(5-36), NPY(4-36), NPY(3-36).Ref: See e.g., Gehlert et al., Proc Soc Exp Biol Med 218: 7-22, 1998;Sheikh et al., Am J Physiol 261: G701-15, November 1991.Internal Deletions, including but not limited to: (1-4)-Aca-(14-36)pNPY,(1-4)-Aca-(15-36)pNPY, (1-4)-Aca-(16-36)pNPY, (1-4)-Aca-(17-36)pNPY, (1-4)-Aca-(18-36)pNPY, (1-4)-(31-36)pNPY11, (1-4)-Aca-(31-36)pNPY, (4-1)-(31-36)pNPY, (4-1)-Aca-(31-36)pNPY, (4-1)_(D)-(31-36)pNPY, (4-1)D-Aca-(31-36)pNPY.Ref: Fournier et al., Mol Pharmacol 45(1): 93-101, January 1994.Additional Internal Deletion Mutants, including but not limited to: des-AA¹⁰⁻¹⁷-NPY, des-AA¹⁰⁻¹⁷, Ac-[D-Lys⁹(ε-Ac-Ala)]NPY, des-AA¹⁰⁻¹⁷, Ac[D-Lys⁹(ε-Ac-Ala)]NPY, des-AA¹⁰⁻¹⁷[Ala^(7,21)]NPY, des-AA¹⁰⁻¹⁷[Cys^(7,21)]NPY, des-AA¹⁰⁻¹⁷[Glu⁷, Lys²¹]NPY, des-AA¹¹⁻¹⁷[D-Lys¹⁰(ε-Ac), Cys^(7,21)]NPY, des-AA¹⁰⁻¹⁷[D-Cys⁷,D-Lys(ε-Ac), Cys²¹]NPY, des-AA¹⁰⁻¹⁷[D-Cys⁷, Lys⁹(ε-Ac), Cys²¹]NPY, des-AA¹⁰⁻¹⁷[Cys^(7,21), Pro³⁴]NPY, des-AA¹⁰⁻¹⁷[Asp⁷, Dpr²¹, Pro³⁴]NPY, des-AA¹⁰⁻¹⁷[Glu⁷, Lys²¹, Pro³⁴]NPY, des-AA¹⁰⁻¹⁷[Cys^(7,21), Leu³¹, Pro³⁴]NPY, des-AA¹⁰⁻²⁰[Cys^(7,21),Pro³⁴]NPY,des-AA¹⁰⁻¹⁷[Cys^(2,27)]NPY, des-AA¹⁰⁻¹⁷[Cys², D-Cys²⁷]NPY. Ref: Kirby et al., J Med Chem 38: 4579-86, 1995.Cyclic agonist of NPY, including but-not limited to: [Lys 25-Glu 29]NPY(Ac-25-36), [Glu 25-Lys 29]NPY(Ac-25-36), [Lys 26-Glu31]NPY(Ac-25-36), [Glu 27-Lys31]NPY(Ac-25-36), [Lys28-Glu 32]NPY(Ac-25-36), [Lys27-Glu34]NPY(Ac-25-36).Ref: Rist et al., Eur J Biochem 247: 1019-1028, 1997.D-amino acid substitutions: [D-Tyr¹]NPY, [D-Pro²]NPY, [D-Ser³]NPY, [D-Lys⁴]NPY, [D-Pro⁵]NPY, [D-Asp⁶]NPY, [D-Asn⁷]NPY, [D-Pro⁸]NPY, [D-Ala⁹]NPY, [D-Glu¹⁰]NPY, [D-Asp¹¹]NPY, [D-Ala¹²]NPY, [D-Pro¹³]NPY, [D-Ala¹⁴]NPY, [D-Glu¹⁵]NPY, [D-Asp¹⁶]NPY, [D-Leu¹⁷]NPY, [D-Ala¹⁸]NPY, [D-Arg¹⁹]NPY, [D-Tyr²⁰]NPY, [D-Tyr²¹]NPY, [D-Ser²²]NPY, [D-Ala²³]NPY, [D-Leu²⁴]NPY, [D-Arg²⁵]NPY, [D-His²⁶]NPY, [D-Tyr²⁷]NPY, [D-Ile²⁸]NPY, [D-Asn²⁹]NPY, [D-Leu³⁰]NPY, [D-Ile³¹]NPY, [D-Thr³²]NPY, [D-Arg³³]NPY, [D-Gln³⁴]NPY, [D-Arg³⁵]NPY, [D-Tyr³⁶]NPY, [D-Tyr¹, D-Pro²]NPY, [D-Ser³, D-Lys⁴]NPY, [D-Pro⁵, D-Asp⁶]NPY, [D-Asn⁷, D-Pro⁸]NPY, [D-Glu¹⁰, D-Asp¹¹]NPY,[D-Asp¹¹, D-Ala¹²]NPY, [D-Pro¹³, D-Ala¹⁴]NPY, [D-Glu¹⁵, D-Asp¹⁶]NPY, [D-Met¹⁷, D-Ala¹⁸]NPY, [D-Arg¹⁹, D-Tyr²⁰]NPY, [D-Tyr²¹, D-Ser²²]NPY, [D-Ala²³, D-Leu²⁴]NPY, [D-Arg²⁵, D-His²⁶]NPY, [D-Tyr²⁷, D-Ile²⁸]NPY, [D-Asn²⁹, D-Leu³⁰]NPY, [D-Ile³¹, D-Thr³²]NPY, [D-Arg³³, D-Gln³⁴]NPY, [D-Arg³⁵, D-Tyr³⁶]NPY.Ref: Kirby et al., J Med Chem 36: 3802-08, 1993; Grundemar et al.,Regulatory Peptides 62: 131-136, 1996. Other NPY Agonist and AnalogsNPY (3-36) SKPDNPGEDAPAEDMARYYSALRHYINLITRQRY (SEQ ID NO: 335)Ref: Grandt et al., Regulatory Peptides 67(1): 33-7, 1996.N-Acetyl NPY(24-36) LRHYINLITRQRY (SEQ ID NO: 213)Ref: Potter et al., Eur J Pharmacol 267(3): 253-262, May 17, 1994.N-Acetyl [Leu²⁸,  LRHYLNLLTRQRY Leu³¹] NPY(24-36) (SEQ ID NO: 214)Ref: Potter et al., Eur J Pharmacol 267(3): 253-262, May 17, 1994.[Leu²⁸, Leu³¹] LRHYLNLLTRQRY NPY(24-36) (SEQ ID NO: 215)Ref: Potter et al., Eur J Pharmacol 267(3): 253-262, May 17, 1994.[Leu¹⁷, Gln¹⁹, Ala²¹, Ala²², PAEDLAQYAAELRHYLNLLTRQRYGlu²³, Leu²⁸, Leu³¹] (SEQ ID NO: 216) NPY(13-36)Ref: Potter et al., Eur J Pharmacol 267(3): 253-262, May 17, 1994.Cyclo S-S [Cys²⁰, Cys²⁴] SKPDNPGEDAPAEDMARCYSACRHYINLITRQRY pNPY(SEQ ID NO: 315)Ref: Soll et al., Eur J Biochem 268(10): 2828-37, May 2001.Cyclo-(28/32)-Ac-[Lys²⁸- RHYLNLIGRQRY Glu³²]-(25-36)-pNPY(SEQ ID NO: 316)Ref: Cabrele et al., J Pept Sci 6(3): 97-122, March 2000.Cyclo-(27/31)-Ac-[Glu²⁷- RHGLNLLGRQRY Lys³¹]-(25-36)-pNPY(SEQ ID NO: 317)Ref: Cabrele et al., J Pept Sci 6(3): 97-122, March 2000.[Tyr³², Leu³⁴]NPY(27-36) YINLIYRLRY (SEQ ID NO: 318)Ref: Leban et al., J Med Chem 38: 1150-57, 1995.[Tyr³², Leu³⁴]NPY(26-36) HYINLIYRLRY (SEQ ID NO: 319)Ref: Leban et al., J Med Chem 38: 1150-57, 1995.[Tyr³², Leu³⁴]NPY(25-36) RHYINLIYRLRY (SED ID NO: 320)Ref: Leban et al., J Med Chem 38: 1150-57, 1995. [Leu³¹]NPY(27-36)YINLLYRQRY (SEQ ID NO: 321)Ref: Leban et al., J Med Chem 38: 1150-57, 1995.[Tyr³², Leu³⁴](1-4)-Ahr- YPSL-Aha-YINLIYRLRY (27-36)NPY (SED ID NO: 322)Ref: Leban et al., J Med Chem 38: 1150-57, 1995.[Tyr³², Leu³⁴]NPY(28-36) INLIYRLRY (SEQ ID NO: 323)Ref: Leban et al., J Med Chem 38: 1150-57, 1995. PP (human)ASLEPEYPGDNATPEQMAQYAAELRRYTNMLTRPRY (SEQ ID NO: 3)Ref: Kimmel et al., Endocrinology 83: 1323-30, 1968. Variations of PPN-Terminal Deletions including but not limited to: PP(26-36),PP(25-36),PP(24-36), PP(23-36), PP(22-36), PP(21-36), PP(20-36),PP(19-36), PP(18-36), PP(17-36), PP(16-36), PP(15-36), PP(14-36),PP(13-36), PP(12-36), PP(11-36), PP(10-36), PP(9-36), PP(8-36),PP(7-36), PP(6-36), PP(5-36), PP(4-36), PP(3-36).

TABLE 3 EXAMPLES OF CONSERVATIVE AMINO ACID SUBSTITUTIONS OF PYY PEPTIDESEQUENCE Single point mutations of  PYY(25-36) [Lys²⁵]PPY(25-36)KHYLNLVTRQRY (SEQ ID NO: 36) [Thr²⁷]PPY(25-36) RHTLNLVTRQRY(SEQ ID NO: 37) [Phe²⁷]PPY(25-36) RHFLNLVTRQRY (SEQ ID NO: 38)[Ile²⁸]PYY (25-36) RHYINLVTRQRY (SEQ ID NO: 39) [Val²⁸]PYY (25-36)RHYVNLVTRQRY (SEQ ID NO: 40) [Gln²⁹]PYY (25-36) RHYLQLVTRQRY(SEQ ID NO: 41) [Ile³⁰]PYY (25-36) RHYLNIVTRQRY (SEQ ID NO: 42)[Val³⁰]PYY (25-36) RHYLNVVTRQRY (SEQ ID NO: 43) [Ile³¹]PYY (25-36)RHYLNLITRQRY (SEQ ID NO: 44) [Leu³¹]PYY (25-36) RHYLNLLTRQRY(SEQ ID NO: 45) [Ser³²]PYY (25-36) RHYLNLVSRQRY (SEQ ID NO: 46)[Lys³³]PYY (25-36) RHYLNLVTKQRY (SEQ ID NO: 47) [Asn³⁴]PYY (25-36)RHYLNLVTRNRY (SEQ ID NO: 48) [Lys³⁵]PYY (25-36) RHYLNLVTRQKY(SEQ ID NO: 49) [Thr³⁶]PYY (25-36) RHYLNLVTRQRT (SEQ ID NO: 50)[Phe³⁶]PYY (25-36) RHYLNLVTRQRF (SEQ ID NO: 51) Double point mutations[Lys²⁵, Thr²⁷]PPY(25-36) KHTLNLVTRQRY (SEQ ID NO: 52)[Lys²⁵, Phe²⁷]PPY(25-36) KHFLNLVTRQRY (SEQ ID NO: 53)[Lys²⁵, Ile²⁸]PPY(25-36) KHYINLVTRQRY (SEQ ID NO: 54)[Lys²⁵, Val²⁸]PPY(25-36) KHYVNLVTRQRY (SEQ ID NO: 55)[Lys²⁵, Gln²⁹]PPY(25-36) KHYLQLVTRQRY (SEQ ID NO: 56)[Lys²⁵, Ile³⁰]PPY(25-36) KHYLNLVTRQRY (SEQ ID NO: 57)[Lys²⁵, Val³⁰]PPY(25-36) KHYLNLVTRQRY (SEQ ID NO: 58)[Lys²⁵, Ile³¹]PPY(25-36) KHYLNLITRQRY (SEQ ID NO: 59)[Lys²⁵, Leu³¹]PPY(25-36) KHYLNLLTRQRY (SEQ ID NO: 60)[Lys²⁵, Ser³²]PPY(25-36) KHYLNLVTRQRY (SEQ ID NO: 61)[Lys²⁵, Lys³³]PPY(25-36) KHYLNLVTKQRY (SEQ ID NO: 62)[Lys²⁵, Asn³⁴]PPY(25-36) KHYLNLVTRNRY (SEQ ID NO: 63)[Lys²⁵, Lys³⁵]PPY(25-36) KHYLNLVTRQKY (SEQ ID NO: 64)[Lys²⁵, Thr³⁶]PPY(25-36) KHYLNLVTRQRT (SEQ ID NO: 65)[Lys²⁵, Phe³⁶]PPY(25-36) KHYLNLVTRQRF (SEQ ID NO: 66)[Thr²⁷, Ile²⁸]PPY(25-36) RHTINLVTRQRY (SEQ ID NO: 67)[Thr²⁷, Val²⁸]PPY(25-36) RHTVNLVTRQRY (SEQ ID NO: 68)[Thr²⁷, Gln²⁹]PPY(25-36) RHTLQLVTRQRY (SEQ ID NO: 69)[Thr²⁷, Ile³⁰]PPY(25-36) RHTLNIVTRQRY (SEQ ID NO: 70)[Thr²⁷, Val³⁰]PPY(25-36) RHTLNVVTRQRY (SEQ ID NO: 71)[Thr²⁷, Ile³¹]PPY(25-36) RHTLNLITRQRY (SEQ ID NO: 72)[Thr²⁷, Leu³¹]PPY(25-36) RHTLNLLTRQRY (SEQ ID NO: 73)[Thr²⁷, Ser³²]PPY(25-36) RHTLNLVSRQRY (SEQ ID NO: 74)[Thr²⁷, Lys³³]PPY(25-36) RHTLNLVTKQRY (SEQ ID NO: 75)[Thr²⁷, Asn³⁴]PPY(25-36) RHTLNLVTRNRY (SEQ ID NO: 76)[Thr²⁷, Lys³⁵]PPY(25-36) RHTLNLVTRQKY (SEQ ID NO: 77)[Thr²⁷, Thr³⁶]PPY(25-36) RHTLNLVTRQRT (SEQ ID NO: 78)[Thr²⁷, Phe³⁶]PPY(25-36) RHTLNLVTRQRF (SEQ ID NO: 79)[Phe²⁷, Ile²⁸]PPY(25-36) RHFINLVTRQRY (SEQ ID NO: 80)[Phe²⁷, Val²⁸]PPY(25-36) RHFVNLVTRQRY (SEQ ID NO: 81)[Phe²⁷, Gln²⁹]PPY(25-36) RHFLQLVTRQRY (SEQ ID NO: 82)[Phe²⁷, Ile³⁰]PPY(25-36) RHFLNIVTRQRY (SEQ ID NO: 83)[Phe²⁷, Val³⁰]PPY(25-36) RHFLNVVTRQRY (SEQ ID NO: 84)[Phe²⁷, Ile³¹]PPY(25-36) RHFLNLITRQRY (SEQ ID NO: 85)[Phe²⁷, Leu³¹]PPY(25-36) RHFLNLLTRQRY (SEQ ID NO: 86)[Phe²⁷, Ser³²]PPY(25-36) RHFLNLVSRQRY (SEQ ID NO: 87)[Phe²⁷, Lys³³]PPY(25-36) RHFLNLVTKQRY (SEQ ID NO: 88)[Phe²⁷, Asn³⁴]PPY(25-36) RHFLNLVTRNRY (SEQ ID NO: 89)[Phe²⁷, Lys³⁵]PPY(25-36) RHFLNLVTRQKY (SEQ ID NO: 90)[Phe²⁷, Thr³⁶]PPY(25-36) RHFLNLVTRQRT (SEQ ID NO: 91)[Phe²⁷, Phe³⁶]PPY(25-36) RHFLNLVTRQRF (SEQ ID NO: 92)[Gln²⁹, Ile³⁰]PYY (25-36) RHYLQIVTRQRY (SEQ ID NO: 93)[Gln²⁹, Val³⁰]PYY (25-36) RHYLQVVTRQRY (SEQ ID NO: 94)[Gln²⁹, Ile³¹]PYY (25-36) RHYLQLITRQRY (SEQ ID NO: 95)[Gln²⁹, Leu³¹]PYY (25-36) RHYLQLLTRQRY (SEQ ID NO: 96)[Gln²⁹, Ser³²]PYY (25-36) RHYLQLVSRQRY (SEQ ID NO: 97)[Gln²⁹, Leu³³]PYY (25-36) RHYLQLVTKQRY (SEQ ID NO: 98)[Gln²⁹, Asn³⁴]PYY (25-36) RHYLQLVTRNRY (SEQ ID NO: 99)[Gln²⁹, Leu³⁵]PYY (25-36) RHYLQLVTRQKY (SEQ ID NO: 100)[Gln²⁹, Thr³⁶]PYY (25-36) RHYLQLVTRQRT (SEQ ID NO: 101)[Gln²⁹, Phe³⁶]PYY (25-36) RHYLQLVTRQRF (SEQ ID NO: 102)[Ile³⁰, Ile³¹]PYY (25-36) RHYLNIITRQRY (SEQ ID NO: 103)[Ile³⁰, Leu³¹]PYY (25-36) RHYLNILTRQRY (SEQ ID NO: 104)[Ile³⁰, Ser³²]PYY (25-36) RHYLNIVSRQRY (SEQ ID NO: 105)[Ile³⁰, Lys³³]PYY (25-36) RHYLNIVTKQRY (SEQ ID NO: 106)[Ile³⁰, Asn³⁴]PYY (25-36) RHYLNIVTRNRY (SEQ ID NO: 107)[Ile³⁰, Lys³⁵]PYY (25-36) RHYLNIVTRQKY (SEQ ID NO: 108)[Ile³⁰, Thr³⁶]PYY (25-36) RHYLNIVTRQRT (SEQ ID NO: 109)[Ile³⁰, Phe³⁶]PYY (25-36) RHYLNIVTRQRF (SEQ ID NO: 110)[Val³⁰, Ile³¹]PYY (25-36) RHYLNVITRQRY (SEQ ID NO: 111)[Val³⁰, Leu³¹]PYY (25-36) RHYLNVLTRQRY (SEQ ID NO: 112)[Val³⁰, Ser³²]PYY (25-36) RHYLNVVSRQRY (SEQ ID NO: 113)[Val³⁰, Lys³³]PYY (25-36) RHYLNVVTKQRY (SEQ ID NO: 114)[Val³⁰, Asn³⁴]PYY (25-36) RHYLNVVTRNRY (SEQ ID NO: 115)[Val³⁰, Lys³⁵]PYY (25-36) RHYLNVVTRQKY (SEQ ID NO: 116)[Val³⁰, Thr³⁶]PYY (25-36) RHYLNVVTRQRT (SEQ ID NO: 117)[Val³⁰, Phe³⁶]PYY (25-36) RHYLNVVTRQRF (SEQ ID NO: 118)[Ile³¹, Ser³²]PYY (25-36) RHYLNLISRQRY (SEQ ID NO: 119)[Ile³¹, Lys³³]PYY (25-36) RHYLNLITKQRY (SEQ ID NO: 120)[Ile³¹, Asn³⁴]PYY (25-36) RHYLNLITRNRY (SEQ ID NO: 121)[Ile³¹, Lys³⁵]PYY (25-36) RHYLNLITRQKY (SEQ ID NO: 122)[Ile³¹, Thr³⁶]PYY (25-36) RHYLNLITRQRT (SEQ ID NO: 123)[Leu³¹, Phe³⁶]PYY (25-36) RHYLNLITRQRF (SEQ ID NO: 124)[Leu³¹, Ser³²]PYY (25-36) RHYLNLLSRQRY (SEQ ID NO: 125)[Val³¹, Lys³³]PYY (25-36) RHYLNLLTKQRY (SEQ ID NO: 126)[Leu³¹, Asn³⁴]PYY (25-36) RHYLNLLTRNRY (SEQ ID NO: 127)[Leu³¹, Lys³⁵]PYY (25-36) RHYLNLLTRQKY (SEQ ID NO: 128)[Leu³¹, Thr³⁶]PYY (25-36) RHYLNLLTRQRT (SEQ ID NO: 129)[Leu³¹, Phe³⁶]PYY (25-36) RHYLNLLTRQRF (SEQ ID NO: 130)[Ser³², Lys³³]PYY (25-36) RHYLNLVSKQRY (SEQ ID NO: 131)[Ser³², Asn³⁴]PYY (25-36) RHYLNLVSRNRY (SEQ ID NO: 132)[Ser³², Lys³⁵]PYY (25-36) RHYLNLVSRQKY (SEQ ID NO: 133)[Ser³², Thr³⁶]PYY (25-36) RHYLNLVSRQRT (SEQ ID NO: 134)[Ser³², Phe³⁶]PYY (25-36) RHYLNLVSRQRY (SEQ ID NO: 135)[Lys³³, Asn³⁴]PYY (25-36) RHYLNLVTKNRY (SEQ ID NO: 136)[Lys³³, Lys³⁵]PYY (25-36) RHYLNLVTKQKY (SEQ ID NO: 137)[Lys³³, Thr³⁶]PYY (25-36) RHYLNLVTKQRT (SEQ ID NO: 138)[Lys³³, Phe³⁶]PYY (25-36) RHYLNLVTKQRF (SEQ ID NO: 139)[Asn³⁴, Lys³⁵]PYY (25-36) RHYLNLVTRNKY (SEQ ID NO: 140)[Asn³⁴, Thr³⁶]PYY (25-36) RHYLNLVTRNRT (SEQ ID NO: 141)[Asn³⁴, Phe³⁶]PYY (25-36) RHYLNLVTRNRF (SEQ ID NO: 142)[Lys³⁵, Thr³⁶]PYY (25-36) RHYLNLVTRQKT (SEQ ID NO: 143)[Lys³⁵, Phe³⁶]PYY (25-36) RHYLNLVTRQKF (SEQ ID NO: 144)Point Mutations of  PYY(24-36) PYY(24-36) LRHYLNLVTRQRY (SEQ ID NO: 145)[Ile²⁴]PYY(24-36) IRHYLNLVTRQRY (SEQ ID NO: 146) [Val²⁴]PYY(24-36)VRHYLNLVTRQRY (SEQ ID NO: 147)

Also included as PYY(24-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of any of thesethree mutants with any of the above listed mutants for PYY(25-36), e.g.,[Lys²⁵]PPY(24-36) (Amino acid sequence=LKHYLNLVTRQRY (SEQ ID NO: 191))would result from combining the mutations from SEQ ID NO: 36 with SEQ IDNO: 145.

Point Mutations of PYY(23-36)

PEPTIDE SEQUENCE PYY(23-36) SLRHYLNLVTRQRY (SEQ ID NO: 148)[Thr²³]PYY(23-36) TLRHYLNLVTRQRY (SEQ ID NO: 149)

Also included as PYY(23-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(24-36), e.g.,[Lys²⁵]PPY(23-36) (Amino acid sequence=SLKHYLNLVTRQRY (SEQ ID NO: 192))would result from combining the mutations from SEQ ID NO: 36 with SEQ IDNO: 148.

Point Mutations of PYY(22-36)

PEPTIDE SEQUENCE PYY(22-36) ASLRHYLNLVTRQRY (SEQ ID NO: 150)[Ser²²)PYY(22-36) SSLRHYLNLVTRQRY (SEQ ID NO: 151)

Also included as PYY(22-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(23-36), e.g.,[Lys²⁵]PPY(22-36) (Amino acid sequence=ASLKHYLNLVTRQRY (SEQ ID NO: 193))would result from combining the mutations from SEQ ID NO: 36 with SEQ IDNO: 150.

Point Mutations of PYY(21-36)

PEPTIDE SEQUENCE PYY(21-36) YASLRHYLNLVTRQRY (SEQ ID NO: 152)[Thr²¹]PYY(21-36) TASLRHYLNLVTRQRY (SEQ ID NO: 153) [Phe²¹]PYY(21-36)FASLRHYLNLVTRQRY (SEQ ID NO: 154)

Also included as PYY(21-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of any of thesethree mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(22-36), e.g.,[Lys²⁵]PPY(21-36) (Amino acid sequence=YASLKHYLNLVTRQRY (SEQ ID NO:194)) would result from combining the mutations from SEQ ID NO: 36 withSEQ ID NO: 152.

Point Mutations of PYY(20-36)

PEPTIDE SEQUENCE PYY(20-36) YYASLRHYLNLVTRQRY (SEQ ID NO: 155)[Thr²⁰]PYY(20-36) TYASLRHYLNLVTRQRY (SEQ ID NO: 156) [Phe²⁰]PYY(20-36)FYASLRHYLNLVTRQRY (SEQ ID NO: 157)

Also included as PYY(20-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of any of thesethree mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(21-36), e.g.,[Lys²⁵]PPY(20-36) (Amino acid sequence=YYASLKHYLNLVTRQRY (SEQ ID NO:195)) would result from combining the mutations from SEQ ID NO: 36 withSEQ ID NO: 155.

Point Mutations of PYY(19-36)

PEPTIDE SEQUENCE PYY(19-36) RYYASLRHYLNLVTRQRY (SEQ ID NO: 158)[Lys¹⁹]PYY(19-36) KYYASLRHYLNLVTRQRY (SEQ ID NO: 159)

Also included as PYY(19-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(20-36), e.g.,[Lys²⁵]PPY(19-36) (Amino acid sequence=RYYASLKHYLNLVTRQRY (SEQ ID NO:196)) would result from combining the mutations from SEQ ID NO: 36 withSEQ ID NO: 158.

Point Mutations of PYY(18-36)

PEPTIDE SEQUENCE PYY(18-36) NRYYASLRHYLNLVTRQRY (SEQ ID NO: 160)[Gln¹⁸]PYY(18-36) QRYYASLRHYLNLVTRQRY (SEQ ID NO: 161)

Also included as PYY(18-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(19-36), e.g.,[Lys²]PPY(18-36) (Amino acid sequence=NRYYASLKHYLNLVTRQRY (SEQ ID NO:197)) would result from combining the mutations from SEQ ID NO: 36 withSEQ ID NO: 160.

Point Mutations of PYY(17-36)

PEPTIDE SEQUENCE PYY(17-36) LNRYYASLRHYLNLVTRQRY (SEQ ID NO: 162)[Ile¹⁷]PYY(17-36) INRYYASLRHYLNLVTRQRY (SEQ ID NO: 163)[Val¹⁷]PYY(17-36) VNRYYASLRHYLNLVTRQRY (SEQ ID NO: 164)

Also included as PYY(17-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of any of thesethree mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(18-36), e.g.,[Lys²⁵]PPY(17-36) (Amino acid sequence=LNRYYASLKHYLNLVTRQRY (SEQ ID NO:198)) would result from combining the mutations from SEQ ID NO: 36 withSEQ ID NO: 162.

Point Mutations of PYY(16-36)

PEPTIDE SEQUENCE PYY(16-36) ELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 165)[Asp¹⁶]PYY(16-36) DLNRYYASLRHYLNLVTRQRY (SEQ ID NO: 166)

Also included as PYY(16-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(17-36), e.g.,[Lys²⁵]PPY(16-36) (Amino acid sequence=ELNRYYASLKHYLNLVTRQRY (SEQ ID NO:199)) would result from combining the mutations from SEQ ID NO: 36 withSEQ ID NO: 165.

Point Mutations of PYY(15-36)

PEPTIDE SEQUENCE PYY(15-36) EELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 167)[Asp¹⁵]PYY(15-36) DELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 168)

Also included as PYY(15-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(16-36), e.g.,[Lys²⁵]PPY(15-36) (Amino acid sequence=EELNRYYASLKHYLNLVTRQRY (SEQ IDNO: 200)) would result from combining the mutations from SEQ ID NO: 36with SEQ ID NO: 167.

Point Mutations of PYY(14-36)

PEPTIDE SEQUENCE PYY(14-36) PEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 169)

Also included as PYY(14-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of thisPYY(14-36) mutant with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(15-36), e.g.,[Lys²⁵]PPY(23-36) (Amino acid sequence=PEELNRYYASLKHYLNLVTRQRY (SEQ IDNO: 201) would result from combining the mutations from SEQ ID NO: 36with SEQ ID NO: 169.

Point Mutations of PYY(13-36)

PEPTIDE SEQUENCE PYY(13-36) SPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 170)[Thr¹³]PYY(13-36) TPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 171)

Also included as PYY(13-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(14-36), e.g.,[Lys²⁵]PPY(13-36) (Amino acid sequence=SEELNRYYASLKHYLNLVTRQRY (SEQ IDNO: 202)) would result from combining the mutations from SEQ ID NO: 36with SEQ ID NO: 170.

Point Mutations of PYY(12-36)

PEPTIDE SEQUENCE PYY(12-36) ASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 172)[Ser¹²]PYY(12-36) ASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 173)

Also included as PYY(12-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(13-36), e.g.,[Lys²⁵]PPY(12-36) (Amino acid sequence=ASEELNRYYASLKHYLNLVTRQRY (SEQ IDNO: 203)) would result from combining the mutations from SEQ ID NO: 36with SEQ ID NO: 172.

Point Mutations of PYY(11-36)

PEPTIDE SEQUENCE PYY(11-36) DASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 174)[Glu¹¹]PYY(11-36) EASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 175)

Also included as PYY(12-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(12-36), e.g.,[Lys²⁵]PPY(11-36) (Amino acid sequence=DASEELNRYYASLKHYLNLVTRQRY (SEQ IDNO: 204)) would result from combining the mutations from SEQ ID NO: 36with SEQ ID NO: 174.

Point Mutations of PYY(10-36)

PEPTIDE SEQUENCE PYY(10-36) EDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 176)[Asp¹⁰]PYY(10-36) DDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 177)

Also included as PYY(10-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(11-36), e.g.,[Lys²⁵]PPY(10-36) (Amino acid sequence=EDASEELNRYYASLKHYLNLVTRQRY (SEQID NO: 205)) would result from combining the mutations from SEQ ID NO:36 with SEQ ID NO: 176.

Point Mutations of PYY(9-36)

PEPTIDE SEQUENCE PYY(9-36) GEDASPEELNRYYASLRHYLNLVTRQRY (SEQ ID NO: 178)

Also included as PYY(9-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of thisPPY(9-36) mutant with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(10-36), e.g.,[Lys²⁵]PPY(9-36) (Amino acid sequence=GEDASPEELNRYYASLKHYLNLVTRQRY (SEQID NO: 206)) would result from combining the mutations from SEQ ID NO:36 with SEQ ID NO: 178.

Point Mutations of PYY(8-36)

PEPTIDE SEQUENCE PYY(8-36) PGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 179)

Also included as PYY(8-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of thisPPY(8-36) mutant with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(9-36), e.g.,[Lys²⁵]PPY(8-36) (Amino acid sequence=SEQ ID NO: 207)) would result fromcombining the mutations from SEQ ID NO: 36 with SEQ ID NO: 179.

Point Mutations of PYY(7-36)

PEPTIDE SEQUENCE PYY(7-36) APGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 180) [Ser⁹]PYY(7-36) SPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 181)

Also included as PYY(7-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(8-36), e.g.,[Lys²⁵]PPY(7-36) (Amino acid sequence=APGEDASEELNRYYASLKHYLNLVTRQRY (SEQID NO: 208)) would result from combining the mutations from SEQ ID NO:36 with SEQ ID NO: 180.

Point Mutations of PYY(6-36)

PEPTIDE SEQUENCE PYY(6-36) EAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 182) [Asp⁶]PYY(6-36) DAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 183)

Also included as PYY(6-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of either ofthese two mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(7-36), e.g.,[Lys²⁵]PPY(6-36) (Amino acid sequence=EAPGEDASEELNRYYASLKHYLNLVTRQRY(SEQ ID NO: 209)) would result from combining the mutations from SEQ IDNO: 36 with SEQ ID NO: 182.

Point Mutations of PYY(5-36)

PEPTIDE SEQUENCE PYY(5-36) PEAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 184)

Also included as PYY(5-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of thisPPY(5-36) mutant with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(6-36), e.g.,[Lys²⁵]PPY(5-36) (Amino acid sequence=PEAPGEDASPEELNRYYASLKHYLNLVTRQRY(SEQ ID NO: 210)) would result from combining the mutations from SEQ IDNO: 36 with SEQ ID NO: 184.

Point Mutations of PYY(4-36)

PEPTIDE SEQUENCE PYY(4-26) KPEAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 185) [Arg⁴]PYY(4-36) RPEAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 186) [Gln⁴]PYY(4-36) QPEAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 187) [Asn⁴]PYY(4-36) NPEAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 188)

Also included as PYY(4-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of any of thesefour mutants with any of the above listed mutants for PYY(25-36), and/orany of the above listed mutants for PYY(5-36), e.g., [Lys²⁵]PPY(4-36)(Amino acid sequence=KPEAPGEDASEELNRYYASLKHYLNLVTRQRY (SEQ ID NO: 211))would result from combining the mutations from SEQ ID NO: 36 with SEQ IDNO: 185.

Point Mutations of PYY(3-36)

PEPTIDE SEQUENCE PYY(3-36) IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 1) [Leu³]PYY(3-36) LKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 189) [Val³]PYY(3-36) VKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY(SEQ ID NO: 190)

Also included as PYY(3-36) mutations are polypeptide variations (aminoacid sequence variations) resulting from the combination of any of thesethree mutants with any of the above listed mutants for PYY(25-36),and/or any of the above listed mutants for PYY(4-36), e.g.,[Lys²⁵]PPY(3-36) (Amino acid sequence=IKPEAPGEDASEELNRYYASLKHYLNLVTRQRY(SEQ ID NO: 212)) would result from combining the mutations from SEQ IDNO: 36 with SEQ ID NO: 1.

Also contemplated are PYY agonists (NPY analogs) having the formula:

X-Q-R₁₉-R₂₀-R₂₁-R₂₂-R₂₃-Leu-R₂₅-R₂₆-R₂₇-R₂₈-R₂₉-R₃₀-R₃₁-R₃₂-Arg-R₃₄-Arg-R₃₆-Y

wherein X is H or C^(a) Me or N^(a) Me or desamino or an acyl grouphaving 7 carbon atoms or less; Q is R₁₇-R₁₈, R₁₈ or desQ; R₁₇ is Met,Arg, Nle, Nva, Leu, Ala or D-Ala; R₁₈ is Ala, Ser, Ile, D-Ala, D-Ser orD-Ile; R₁₉ is Arg, Lys or Gln; R₂₀ is Tyr or Phe; R₂₁ is Tyr, Glu, H isor Ala; R₂₂ is Ser, Ala, Thr, Asn or Asp; R₂₃ is Ala, Asp, Glu, Gln, Asnor Ser; R₂₅ is Arg or Gln; R₂₆ is H is, Arg or Gln; R₂₇ is Phe or Tyr;R₂₈ is Ile, Leu, Val or Arg; R₂₉ is Asn or Ile; R₃₀ is Leu, Met, Thr orVal; R₃₁ is Ile, Val or Leu; R₃₂ is Thr or Phe; R₃₄ is Gln, Pro or H is;R₃₆ is Phe or Tyr; and Y is NH₂ or OH; provided that when Q is R₁₈, thenat least one of R₂₇ and R₃₆ is Phe. Analogs of NPY have the followingapplications: potent postsynaptic treatment of hypertension andcardiogenic shock, the treatment of acute cardiovascular circulatoryfailure, and the elevation of intracellular calcium. See U.S. Pat. No.5,026,685.

Certain preferred NPY analogs have the formula:X-R₁₈-Arg-Tyr-Tyr-R₂₂-R₂₃-Leu-Arg-His-Tyr-R₂₈-Asn-Leu-R₃₁-Thr-Arg-Gln-Arg-Tyr-NH₂,wherein X is H or C^(a) Me or N^(a) Me or desamino or an acyl grouphaving 7 carbon atoms or less; R₁₈ is Ala or Ser; R₂₂ is Ser or Ala; R₂₃is Ala or Ser; R₂₇ is Phe or Tyr; R₂₈ is Ile or Leu; R₃₁ is Ile or Val;and R₃₆ is Phe or Tyr; provided that at least one of R₂₇ and R₃₆ is Phe.See U.S. Pat. No. 5,026,685.

Other contemplated NPY analogs have the formula:

X-R₁₇-R₁₈-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-R₂₇-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-R₃₆-NH₂,

wherein R₁₇ is Arg or Leu and R₁₈ is Ser or Ala or Ile; and wherein X,Rn and R₃₆ are as previously indicated.

Still other preferred NPY analogs have the formula:

X-R₁₈-Arg-Tyr-Tyr-Ala-Ser-Leu-R₂₅-His-R₂₇-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-R₃₆-NH₂,

wherein X is desamino or C^(a) Me or N^(a) Me and wherein R₁₈, R₂₅, R₂₇and R₃₆ are as previously indicated.

Examples of such NPY agonists include:

pNPY (17-36) having the formula:

(SEQ ID NO: 217) H-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide hNPY (17-36) having the formula:

(SEQ ID NO: 218) H-Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Phe²⁷]-NPY (18-36) having the formula:

(SEQ ID NO: 219) H-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Phe-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Ac-D-Ala¹⁷]-NPY (17-36) having the formula:

(SEQ ID NO: 220) Ac-D-Ala-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide NPY (19-36) having the formula:

(SEQ ID NO: 221) H-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Nle¹⁷]-NPY (17-36) having the formula:

(SEQ ID NO: 222) H-Nle-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [D-Ser¹⁸]-NPY (18-36) having the formula:

(SEQ ID NO: 223) H-D-Ser-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Ala¹⁷, His²¹]-NPY (17-36) having the formula:

(SEQ ID NO: 224) H-Ala-Ala-Arg-Tyr-His-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [D-Ile¹⁸]-NPY (18-36) having the formula:

(SEQ ID NO: 225) D-Ile-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Ac-Arg¹⁷]-NPY (17-36) having the formula:

(SEQ ID NO: 226) Ac-Arg-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Gln¹⁹]-NPY (19-36) having the formula:

(SEQ ID NO: 227) H-Gln-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Phe²⁰]-NpY (18-36) having the formula:

(SEQ ID NO: 228) H-Ala-Arg-Phe-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [C^(a) MeLeu¹⁷]-pNPY (17-36) having the formula:

(SEQ ID NO: 229) H-Ca MeLeu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [N^(a) MeLeu¹⁷]-pNPY (17-36) having the formula:

(SEQ ID NO: 230) H-N^(a) MeLeu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln- Arg-Tyr-NH₂

The peptide [desamino Ala¹⁸]-NpY (18-36) having the formula:

(SEQ ID NO: 231) desamino-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr- NH₂

The peptide [For-Ala¹⁸, Glu²³, Arg²⁶]-NPY (18-36) having the formula:

(SEQ ID NO: 232) For-Ala-Arg-Tyr-Tyr-Ser-Glu-Leu-Arg-Arg-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Nva¹⁷, Ala²¹, Leu²⁸]-NPY (17-36) having the formula:

(SEQ ID NO: 233) H-Nva-Ala-Arg-Tyr-Ala-Ser-Ala-Leu-Arg-His-Tyr-Leu-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Thr²², Gln²³]-NPY (18-36) having the formula:

(SEQ ID NO: 234) H-Ala-Arg-Tyr-Tyr-Thr-Gln-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [desamino Leu¹⁷, Asn²³, Val³⁰]-NPY (17-36) having theformula:

(SEQ ID NO: 235) H-desamino Leu-Ala-Arg-Tyr-Tyr-Ser-Asn-Leu-Arg-His-Tyr-Ile-Asn-Val-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Asp²², Ser²³, Thr³⁰]-NPY (18-36) having the formula:

(SEQ ID NO: 236) H-Ala-Arg-Tyr-Tyr-Asp-Ser-Leu-Arg-His-Tyr-Ile-Asn-Thr-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Gln²⁵, Leu³¹, Pro³⁴]-NPY (18-36) having the formula:

(SEQ ID NO: 237) H-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Gln-His-Tyr-lle-Asn-Leu-Leu-Thr-Arg-Pro-Arg-Tyr-NH₂

The peptide [Gln²Phe³⁶]-NPY (17-36) having the formula:

(SEQ ID NO: 238) H-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-Gln-Tyr-Arg-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Phe-NH₂

The peptide [Phe³⁶]-pPYY (19-36) having the formula:

(SEQ ID NO: 239) H-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Phe-NH₂

The peptide pPYY (18-36) having the formula:

(SEQ ID NO: 240) H-Ser-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Ac-Ser¹⁸, Phe²⁷]-pPYY (18-36) having the formula:

(SEQ ID NO: 241) Ac-Ser-Arg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Nle¹⁷, Asn²², Phe²⁷]-NPY (17-36) having the formula:

(SEQ ID NO: 242) H-Nle-Ala-Arg-Tyr-Tyr-Asn-Ala-Leu-Arg-His-Phe-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [D-Ala¹⁸, Glu²¹, His³⁴]-NPY (18-36) having the formula:

(SEQ ID NO: 243) H-D-Ala-Arg-Tyr-Glu-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-His-Arg-Tyr-NH₂

The peptide [Bz-Leu¹⁷, Pro³⁴, Phe³⁶]-pNPY (17-36) having the formula:

(SEQ ID NO: 244) Bz-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Pro-Arg-Phe-NH₂

The peptide [Lys¹⁹, Phe²⁷, Val²⁸]-NpY (18-36) having the formula:

(SEQ ID NO: 245) H-Ala-Lys-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Phe-Val-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [D-Ala¹⁷, Val²⁸, Phe³²]-NPY (17-36) having the formula:

(SEQ ID NO: 246) D-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Val-Asn-Leu-Ile-Phe-Arg-Gln-Arg-Tyr-NH₂

The peptide [C^(a) MeSer¹⁸, Met³⁰, Phe³⁶]-NPY (18-36) having theformula:

(SEQ ID NO: 247) H-C^(a) MeSer-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Met-Ile-Thr-Arg-Gln-Arg-Phe-NH₂

The peptide [Arg¹⁷, Ile¹⁸, Phe^(27,36)]-NPY (17-36) having the formula:

(SEQ ID NO: 248) H-Arg-Ile-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Phe-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Phe-NH₂

The peptide [Ser¹⁸, Phe²⁷]-pNPY (17-36) having the formula:

(SEQ ID NO: 249) H-Leu-Ser-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Phe-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [N^(a) MeIle¹⁸. Gln²⁵, Phe²⁷]-NPY (18-36) having theformula:

(SEQ ID NO: 250) N^(a) MeIle-Arg-Tyr-Tyr-Ser-Ala-Leu-Gln-His-Phe-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [D-Ser¹⁸, Phe³⁶]-NPY (18-36) having the formula:

(SEQ ID NO: 251) H-D-Ser-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Phe-NH₂

The peptide [Asp²³, Arg²⁶]hNPY (17-36) having the formula:

(SEQ ID NO: 252) H-Met-Ala-Arg-Tyr-Tyr-Ser-Asp-Leu-Arg-Arg-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [Glu²³, Ile²⁹]-NPY (18-36) having the formula:

(SEQ ID NO: 253) H-Ala-Arg-Tyr-Tyr-Ser-Glu-Leu-Arg-His-Tyr-Ile-Ile-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH₂

The peptide [D-Ala¹⁷]-NPY(17-36)-OH having the formula:

(SEQ ID NO: 254) D-Ala-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-OH.

Other peptide YY agonists have the formula:

wherein:

X is a chain of 0-5 amino acids, inclusive, the N-terminal one of whichis bonded to R₁ and R₂

Y is a chain of 0-4 amino acids, inclusive, the C-terminal one of whichis bonded to R₃ and R₄

R₁ is H, C₁-C₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,napthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl);

R₂ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl);

A²² is an aromatic amino acid, Ala, Aib, Anb, N-Me-Ala, or is deleted;

A²³ is Ser, Thr, Ala, N-Me-Ser, N-Me-Thr, N-Me-Ala, or is deleted;

A²⁴ is Leu, lie, Vat, Trp, Gly, Aib, Anb, N-Me-Leu, or is deleted;

A²⁵ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or an aryl group), Orn,or is deleted;

A²⁶ is His, Thr, 3-Me-His, 1-Me-His, β-pyrozolylalanine, N-Me-His, Arg,Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, a branched orstraight chain C₁-C₁₀ alkyl group, or an aryl group), Orn, or isdeleted;

A²⁷ is an aromatic amino acid other than Tyr;

A²⁸ is Leu, Ile, Vat, Trp, Aib, Aib, Anb, or N-Me-Leu;

A²⁹ is Asn, Ala, Gln, Gly, Trp, or N-Me-Asn;

A³⁰ is Leu, Ile, Val, Trp, Aib, Anb, or N-Me-Leu;

A³¹ is Vat, Ile, Trp, Aib, Anb, or N-Me-Val;

A³² is Thr, Ser, N-Me-Set, or N-Me-Thr;

R₃ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl);

R₄ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl),or a pharmaceutically acceptable salt thereof. See U.S. Pat. No.5,574,010.

Particularly preferred agonists of this formula to be used in the methodof the disclosure include:

(SEQ ID NO: 255) N-α-Ala-Ser-Leu-Arg-His-Trp-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂.

Other peptide YY agonists have the formula:

wherein:

the N-terminal amino acid bonds to R₁ and R₂;

Y is a chain of 0-4 amino acids, inclusive the C-terminal one of whichbonds to R₃ and R₄;

R₁ is H, C₁-C₁₂ alkyl, C₆-C₁₈ aryl, C₁-C₁₂ acyl, C₇-C₁₈ aralkyl, orC₇-C₁₈ alkaryl;

R₂ is H, C₁-C₁₂ alkyl, C₆-C₁₈ aryl, C₁-C₁₂ acyl, C₇-C₁₈ aralkyl, orC₇-C₁₈ alkaryl;

A²⁵ is Mg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or an aryl group), Orn,or is deleted;

A²⁶ is Ala, His, Thr, 3-Me-His, 1-Me-His, β-pyrozolylalanine, N-Me-His,Mg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or an aryl group), Orn oris deleted;

A²⁷ is an aromatic amino acid;

A²⁰ is Leu, Ile, Val, Trp, Aib, Anb, or N-Me-Leu;

A²⁹ is Asn, Ala, Gln, Gly, Trp, or N-Me-Asn;

A³⁰ is Leu, Ile, Val, Trp, Aib, Anb, or N-Me-Leu;

A³¹ is Val, Ile, Tip, Aib, Anb, or N-Me-Val;

A³² is Thr, Set, N-Me-Set, or N-Me-Thr or D-Trp;

R₃ is H, C₁-C₁₂ alkyl, C₆-C₁₈ aryl, C₁-C₁₂ acyl, C₇-C₁₈ aralkyl, orC₇-C₁₈ alkaryl; and

R₄ is H, C₁-C₁₂ alkyl, C₆-C₁₈ aryl, C₁-C₁₂ acyl, C₇-C₁₈ aralkyl, orC₇-C₁₈ alkaryl, or a pharmaceutically acceptable salt thereof. Notethat, unless indicated otherwise, for all peptide YY agonists describedherein, each amino acid residue, e.g., Leu and A¹, represents thestructure of NH—C(R)H—CO—, in which R is the side chain. Lines betweenamino acid residues represent peptide bonds which join the amino acids.Also, where the amino acid residue is optically active, it is the L-formconfiguration that is intended unless D-form is expressly designated.

Other PYY agonists have the formula:

wherein:

X is a chain of 0-5 amino acids, inclusive, the N-terminal one of whichis bonded to R₁ and R₂;

Y is a chain of 0-4 amino acids, inclusive, the C-terminal one of whichis bonded to R₃ and R₄;

R₁ is H, C₁-C₁₂ alkyl (e.g. methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl);

R₂ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl);

A²² is an aromatic amino acid, Ala, Aib, Anb, N-Me-Ala, or is deleted;

A²³ is Ser, Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N Me-Ala, or is deleted;

A²⁴ is leu, Ile, Val, Trp, Gly, Nle, Nva, Aib, Anb, N-Me-Leu, or isdeleted;

A²⁵ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lye-e-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or an aryl group), Orn,or is deleted;

A²⁶ is Ala, His, Thr, 3-Me-His, 1-Me-His, β-pyrozolylalanine, N-Me-His,Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl groups or an aryl group), Orn,or is deleted;

A²⁷ is an aromatic amino acid other than Tyr;

A²⁸ is Leu, Ile, Val, Tip, Nle, Nva, Aib, Anb, or N-Me-Leu;

A²⁹ is Asn, Ala, Gin, Gly, Tip, or N-Me-Asn;

A³⁰ is Leu, Ile, Val, Tip, Nle, Nva, Aib, Anb, or N-Me-Leu;

A³¹ is Val, Leu, Ile, Tip, Nle, Nva, Aib, Anb, or N-Me-Val;

A³² is Thr, Ser, N-Me-Ser, N-Me-Thr, or D-Trp;

R₃ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl);and

R₄ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl),or a pharmaceutically acceptable salt thereof.

In preferred embodiments, A²⁷ is Phe, Nal, Bip, Pcp, Tic, Trp, Bth, Thi,or Dip.

In preferred embodiments X is A¹⁷-A¹⁸-A¹⁹-A²⁰-A²¹ wherein

A¹⁷ is Cys, Leu, Ile, Val, Nle, Nva, Aib, Anb, or N-Me-Leu;

A¹⁸ is Cys, Ser, Thr, N-Me-Ser, or N-Me-Thr;

A¹⁹ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or C₆-C₁₈ aryl group),Cys, or Orn;

A²⁰ is an aromatic amino acid, or Cys; and

A²¹ is an aromatic amino acid, Cys, or a pharmaceutically acceptablesalt thereof. In yet other preferred embodiments, Y is A³³-A³⁴-A³⁵-A³⁶wherein

A³³ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or an aryl group), Cys,or Orn;

A³⁴ is Cys, Gln, Asn, Ala, Gly, Aib, or Anb;

A³⁵ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or C₆-C₁₈ aryl group),Cys, or Orn; and

A³⁶ is an aromatic amino acid, Cys or a pharmaceutically acceptable saltthereof. See U.S. Pat. No. 5,604,203.

Particular embodiments include compounds has the formula:N-α-Ac-Ala-Ser-Leu-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gin-Arg-Tyr-NH₂(SEQ. ID. NO: 325),H-Ala-Ser-Leu-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂ (SEQ.ID. NO: 326), N-α-Ac-Ala-Ser-Leu-Arg-Thr-Arg-Gin-Arg-Tyr-NH₂ (SEQ. ID.NO: 327),N-α-Ac-Ala-Ser-Leu-Arg-His-Thi-Leu-Asn-Leu-Val-Thr-Arg-Gin-Arg-Tyr-NH₂(SEQ. ID. NO: 328),N-α-Ac-Tyr-Ser-Leu-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gin-Arg-Tyr-NH₂(SEQ. ID. NO: 329) or a pharmaceutically acceptable salt thereof.

Other PYY agonists have the formula:

wherein the N-terminal amino acid is bounded to R₁ and R₂; Y is a chainof 0-4 amino acids, inclusive the C-terminal one of which is bonded toR₃ and R₄;

R₁ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl);

R₂ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl);

A²⁵ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or an aryl group), Orn,or is deleted;

A²⁶ is Ala, His, Thr, 3-Me-His, 1-Me-His, β-pyrozolylalanine, N-Me-His,Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl groups or an aryl group), Orn,or is deleted;

A²⁷ is an aromatic amino acid;

A²⁸ is Leu, Ile, Val, Tip, Nle, Nva, Aib, Anb, or N-Me-Leu;

A²⁹ is Asn, Ala, Gin, Gly, Tip, or N-Me-Asn;

A³⁹ is Leu, Ile, Val, Tip, Nle, Nva, Aib, Anb, or N-Me-Leu;

A³¹ is Val, Ile, Tip, Nie, Nva, Aib, Anb, or N-Me-Val;

A³² is Thr, Ser, N-Me-Ser, N-Me-Thr, or D-Trp;

R₃ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl);and

R₄ is H, C₁-C₁₂ alkyl (e.g., methyl), C₆-C₁₈ aryl (e.g., phenyl,naphthaleneacetyl), C₁-C₁₂ acyl (e.g., formyl, acetyl, and myristoyl),C₇-C₁₈ aralkyl (e.g., benzyl), or C₇-C₁₈ alkaryl (e.g., p-methylphenyl),or a pharmaceutically acceptable salt thereof. See U.S. Pat. No.5,604,203.

In particular embodiments, A²⁷ is Phe, Nal, Bip, Pcp, Tic, Trp, Bth,Thi, or Dip.

In particular embodiments X is A³³-A³⁴-A³⁵-A³⁶ wherein

A³³ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or C₆-C₁₈ aryl group),Cys, or Orn;

A³⁴ is Gln, Asn, Ala, Gly, N-Me-Gin, Aib, Cys, or Anb;

A³⁵ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R (where R is H, abranched or straight chain C₁-C₁₀ alkyl group, or C₆-C₁₈ aryl group),Cys, or Orn; and

A³⁶ is an aromatic amino acid, Cys, or a pharmaceutically acceptablesalt thereof.

Preferably, the compound has the formula:N-α-Ac-Arg-His-Phe-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH₂ (SEQ. ID. NO:324).

Examplary PYY agonists include:

YPAKEAPGEDASPEELSTYYASLR (SEQ ID NO: 256) [im-DNP-His²⁶] YLNLVTRZRY-NH₂PYY(22-36) ASLRHYLNLVTRQRY-NH₂ (SEQ ID NO: 257) [Ala³²]PYYASLRHYLNLV[Ala]RQRY-NH₂ (SEQ ID NO: 258) [Ala^(23,32)]PYYA[Ala]LRHYLNLV[Ala]RQRY-NN₂ (SEQ ID NO: 259) [Glu²⁸]PYY(22-36)ASLRHY[Glu]NLVTRQRY-NH₂ (SEQ ID NO: 260) N-α-Ac-PYY(22-36)N-α-Ac-ASLRHYLNLVTRORY-NH₂ (SEQ ID NO: 261) N-α-Ac[p.CL.Phe²⁶]PYYN-α-Ac-ASLR[p.CL.Phe²⁶]YLNLVTRQR (SEQ ID NO: 262) Y-NH₂ N-α-Ac[Glu²⁸]PYYN-α-Ac-ASLRHY[Glu]NLVTRQRY-NH₂ (SEQ ID NO: 263) N-α-Ac[Phe²⁷]PYYN-α-Ac-ASLRH[Phe]ENLVTRQR[N-Me- (SEQ ID NO: 264) Tyr]-NH₂N-α-Ac]8N-Me-Tyr³⁶]PYY N-α-Ac-ASLRHYENLVTR0R[N-Me-Tyr]- (SEQ ID NO: 265)NH₂ N-α-myristoyl-PYY(2214 36) N-α-myristoyl-ASLRHYLNLVTRQRY-NH₂(SEQ ID NO: 266) N-α-naphthateneacetyl-PYY (22-36)N-α-naphthateneacetyl-ASLRHYLNLVT (SEQ ID NO: 267) RQRY-NH₂N-α-Ac[Phe²⁷]PYY N-α-Ac-ASLRH[Phe]ENLVTR0R[N-Me- (SEQ ID NO: 268)Tyr]-NH₂ N-α-Ac-PYY(22-36) N-α-Ac-ASLRHYLNLVTRQRY-NH₂ (SEQ ID NO: 269)N-α-Ac-[Bth²⁷]PYY (22-36) N-α-Ac-ASLRH[Bth]LNLVTRQRY-NH₂(SEQ ID NO: 270) N-α-Ac-[Bip²⁷]PYY (22-36) (SEQ ID NO: 271)N-α-Ac-ASLRH[Bth]LNLVTRQRY-NH₂ (SEQ ID NO: 272)N-α-Ac-[Nal²⁷]PYY (22-36) N-α-Ac-ASLRH[Bth]LNLVTRQRY-NH₂(SEQ ID NO: 273) N-α-Ac-[Trp²⁷]PYY(22-36) (SEQ ID NO: 274)N-α-Ac-ASLRH[Trp]LNLVTRQRY-NH₂ (SEQ ID NO: 275)N-α-Ac-[Thi²⁷]PYY (22-36) N-α-Ac-ASLRN[Thi]LNLVTRQRY-NH₂(SEQ ID NO: 276) N-α-Ac-[Tic²⁷]PYY (22-36)N-α-Ac-ASLRH[Tic]LNLVTRQRY-NH₂ (SEQ ID NO: 277)N-α-Ac-[Phe²⁷]PYY (25-36) N-α-Ac-H[Phe]LNLVTRQRY-NH₂ (SEQ ID NO: 279)N-α-Ac-[Phe²⁷, Thi²⁷]PYY (22-36) N-α-Ac-ASLRH[Phe]LNLVTRQR[Thi]-(SEQ ID NO: 280) NH₂ N-α-Ac-[Thz²⁶, Phe²⁷]PYY (22-36)N-α-Ac-ASLRH[Thz][Phe]LNLVTRQRY- (SEQ ID NO: 281) NH₂N-α-Ac-[Phe²⁷]PYY (22-36) N-α-Ac-[Thz][Phe]LNLVTYRQRY-NH₂(SEQ ID NO: 282) N-α-Ac-[Phe²⁷]PYY (22-36)N-α-Ac-[Phe]SLRN[Phe]LNLVTRQRY- (SEQ ID NO: 289) NH₂N-α-Ac-[Tyr²², Phe²⁷]PYY (22-36) N-α-Ac-[Tyr]SLRH[Phe]LNLVTRQRY-(SEQ ID NO: 290) NH₂ N-α-Ac-[Trp²⁸]PYY (22-36)N-α-Ac-ASLRHY[Trp]NLVTRQRY-NH₂ (SEQ ID NO: 291)N-α-Ac-[Trp²⁸]PYY (22-36) N-α-Ac-ASLRHYLN[Trp]VTRQRY-NH₂(SEQ ID NO: 292) N-α-Ac-[Ala²⁶, Phe²⁷]PYY (22-36)N-α-Ac-ASLR[Ala][Phe]LNLVTRQRY- (SEQ ID NO: 293) NH₂N-α-Ac-[Bth²⁷]PYY (22-36) N-α-Ac-ASLR[Bth]LNLVTRQRY-NH₂ (SEQ ID NO: 294)N-α-Ac-[Phe²⁷]PYY (22-36) N-α-Ac-ASLRH[Phe]LNLVTRQRY-NH₂(SEQ ID NO: 295) N-α-Ac-[Phe^(27,36)]PYY (22-36)N-α-Ac-ASLRH[Phe]LNLVTRQR[Phe]- (SEQ ID NO: 296) NH₂N-α-Ac-[Phe²⁷, D-Trp³²]PYY (22-36) N-α-Ac-ASLRH[Phe]LNLV[D-Trp](SEQ ID NO: 297) RQRY-NH₂

Other PYY agonists include neurophilic Y Y2 receptor specific peptideshaving the formula:

X1(-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13- X14)_(n)-X15

wherein

X1 is NH, CH₃CO or one or two naturally occurring amino acids.

X2 is Leu, Ile or Val.

X3 is Arg, Lys or His.

X4 is His, Lys or Arg.

X5 is Tyr or Phe.

X6 is Leu, Ile or Val.

X7 is Asn or Gln.

X8 is Leu, Ile or Val.

X9 is Leu, Ile or Val.

X10 is Thr or Ser.

X11 is Arg, His or Lys.

X12 is Gln or Asn.

X13 is Arg, His or Lys.

X14 is Tyr or Phe.

X15 is COOH, NH₂ or one or two naturally occurring amino acids with theterminal amino acid being in the normal or carboxamide form; and

n is 1 to 5. See U.S. Pat. No. 5,696,093.

Examplary agonists include:

CH₃CO-L-R-H-Y-L-N-L-L-T-R-Q-R-Y-NH₂ (SEQ ID NO: 298)CH₃CO-L-R-H-Y-I-N-L-I-T-R-Q-R-Y-NH₂ (SEQ ID NO: 299)NH₂-L-R-H-Y-L-N-L-L-T-R-Q-R-Y-NH₂ (SEQ ID NO: 300)NH₂-L-R-H-Y-I-N-L-I-T-R-Q-R-Y-NH₂ (SEQ ID NO: 301)

Other PYY agonists have the formula:

N-α-R¹-[Nle^(24,28,30), Trp²⁷, Nva³¹, ψ^(35/36)]PYY(22-36)-NH₂,

N-α-R¹-[Nle^(24,28), Trp^(27,30), Nva³¹, ψ^(35/36)]PYY(22-36)-NH₂,

N-α-R¹-[Nle^(24,28,30), Phe²⁷, Nva³¹, ψ^(35/36)]PYY(22-36)-NH₂,

N-α-R¹-[Nle^(24,28,30), Phe²⁷, Trp³⁰, Nva³¹, ψ^(35/36)]PYY(22-36)-NH₂,

N-α-R¹-[Trp³⁰, ψ^(35/36)]PYY(25-36)-NH₂,

N-α-R¹-[Trp³⁰]PYY(25-36)-NH₂,

N-α-R¹-[Nle^(24,28), Trp³⁰, Nva³¹, ψ^(35/36)]PYY(22-36)-NH₂ and

N-α-R¹-[Nle²⁸, Trp³⁰, Nva³¹, ψ^(35/36)]PYY(22-36)-NH₂ or apharmaceutically-acceptable salt thereof,

wherein R¹ is H, (C₁-C₁₂)alkyl or (C₁-C₁₂)acyl; and ψ is a pseudopeptidebond selected from the group consisting of —CH₂—NH—, —CH₂—S—, —CH₂—CH₂—,—CH₂—O— and —CH₂—CO—. See U.S. Pat. No. 6,046,162.

Particular compounds of the immediately foregoing group of compounds arewhere R¹ is acetyl and ψ is —CH₂—NH—.

A particular group of compounds is selected from a group consisting of

-   -   N-α-Ac-[Nle^(24,28,30), Trp²⁷, Nva³¹, ψ^(35/36)]PYY(22-36)-NH₂,        (SEQ ID NO: 302)    -   N-α-Ac-[Nle^(24,28), Trp^(27,30), Nva³¹,        ψ^(35/36)]PYY(22-36)-NH₂, (SEQ ID NO: 303)    -   N-α-Ac-[Nle^(24,28,30), Phe²⁷, Nva³¹, ψ^(35/36)]PYY(22-36)-NH₂,        (SEQ ID NO: 304)    -   N-α-Ac-[Nle^(24,28), Phe²⁷, Trp³⁰, Nva³¹,        ψ^(35/36)]PYY(22-36)-NH₂, (SEQ ID NO: 305)

N-α-Ac-[Trp³⁰, ψ^(35/36)]PYY(25-36)-NH₂, (SEQ ID NO: 306)

N-α-Ac-[Trp³⁰]PYY(25-36)-NH₂ (SEQ ID NO: 307) and

N-α-Ac-[Nle²⁸, Trp³⁰, Nva³¹, ψ^(35/36)]PYY(22-36)-NH₂, (SEQ ID NO: 308)or a pharmaceutically acceptable salt thereof.

Another particular compound has the formula N-α-Ac-[Nle^(24,28), Trp³⁰,Nva.sup.³¹, ψ^(35/36)]PYY(22-36)-NH₂ (SEQ. ID. NO: 309) or apharmaceutically acceptable salt thereof.

Another PYY agonist has the formula (A),

having one or two pseudopeptide bonds where each pseudopeptide bond isindependently selected from the group consisting of —CH₂—NH—, —CH₂—S—,—CH₂—CH₂—, —CH₂—O—and —CH₂—CO—; wherein:

R¹⁰ is a chain of 0-5 amino acids, inclusive, where the N-terminal aminoacid is bonded to R¹ and R² by the side chain of the N-terminal aminoacid or by the nitrogen of the amino group of the N-terminal amino acid;

R²⁰ is a chain of 0-4 amino acids, inclusive, where the C-terminal aminoacid is bonded to R³ and R⁴ by the side chain of the C-terminal aminoacid or by the carbon of the carboxyl group of the C-terminal aminoacid;

R¹, R², R³ and R⁴ are each independently selected from the groupconsisting of H, (C₁-C₁₂)alkyl, (C₆-C₁₈)aryl, (C₁-C₁₂)acyl,phenyl(C₁-C₁₂)alkyl and ((C₁-C₁₂)alkyl)₁₋₅-phenyl;

A²² is an aromatic amino acid, Ala, Aib, Anb, N-Me-Ala or is deleted;A²³ is Ser, Thr, Ala, N-Me-Ser, N-Me-Thr, N-Me-Ala or is deleted;

A²⁴ is Leu, Ile, Nle, Val, Trp, Gly, Aib, Anb, N-Me-Leu or is deleted;A²⁵ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-p.epsilon.-NH-Z, Orn oris deleted;

A²⁶ is His, Thr, 3-Me-His, 1-Me-His, β-pyrazolylalanine, N-Me-His, Arg,Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-Z, Orn or is deleted;

A²⁸ is Leu, Ile, Nle, Val, Trp, Aib, Anb or N-Me-Leu;

A²⁹ is Asn, Ala, Gln, Gly, Trp or N-Me-Asn;

A³⁰ is Leu, Ile, Nle, Fla, Val, Trp, Aib, Anb or N-Me-Leu;

A³¹ is Val, Nva, Ile, Tip, Aib, Anb or N-Me-Val; and

A³² is Thr, Ser, N-Me-Ser or N-Me-Thr;

where Z for each occurrence is independently selected from the groupconsisting of H, (C₁-C₁₀)alkyl and (C₆-C₁₈)aryl; or a pharmaceuticallyacceptable salt thereof. See U.S. Pat. No. 6,046,167.

A particular group of compounds of the immediately foregoing group ofcompounds is where R¹⁰ is A¹⁷-A¹⁸-A¹⁹-A²⁰-A²¹;

where A¹⁷ is Cys, Leu, Ile, Val, Nle, Nva, Aib, Anb or N-Me-Leu;

A¹⁸ is Cys, Ser, Thr, N-Me-Ser or N-Me-Thr;

A¹⁹ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R.sup.5, Cys orOrn;

A²⁰ is an aromatic amino acid or Cys;

A²¹ is an aromatic amino acid or Cys;

R²⁰ is A³³-A³⁴-A³⁵-A³⁶,

A³³ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R⁵, Cys or Orn;

A³⁴ is Cys, Gin, Asn, Ala, Gly, N-Me-Gln, Aib or Anb;

A³⁵ is Arg, Lys, homo-Arg, diethyl-homo-Arg, Lys-ε-NH-R⁵, Cys or Orn;

and

A³⁶ is an aromatic amino acid or Cys;

where R⁵ for each occurrence is independently selected from the groupconsisting of H₁ (C₁-C₁₀)alkyl and (C₆-C₁₈) aryl.

A particular group of compounds of the foregoing group of compounds arethe compounds of the formula N-α-Ac-[Fla²⁷)]PYY(25-36)-NH₂ andN-α-Ac[Fla²⁷]P′YY(22-36)-NH₂ or a pharmaceutically acceptable saltthereof.

Another group of PYY agonist has the formula:

or a pharmaceutically acceptable salt thereof wherein

represents an optional bond between the amino acids shown connectedwhere each bond is independently selected from the group consisting of—S—S— only when the amino acids connected are Cys-Cys, —CO—NH—, —CH₂—NH—and

provided that when the optional bond is

it replaces the two amino acids that the optional bond is attached to; qis 1-4; m is 1 to 4;

R³⁰ is OH or —O-R¹, provided that when A¹ to A⁷ are deleted then R³⁰ isalso NH-R¹, where R³⁰ is attached to the carbon atom of the carboxyl ofthe C-terminal amino acid;

R¹ and R² for each occurrence are each independently selected from thegroup consisting of H, (C₁-C₁₂)alkyl, (C₆-C₁₈)aryl, (C₁-C₁₂)acyl,phenyl(C₁-C₁₂)allyl and ((C₁-C₁₂)alkyl)₁₋₅-phenyl where R¹ and R² areattached to the nitrogen of the amine of the N-terminal amino acid;

A¹ is deleted or D- or L- of the following amino acids: Trp, Tyr, Fla,Bth, Nal, Tic, Tic-OH, Dip, Bip or optionally substituted Phe where thePhe is optionally substituted with one to five substituents selectedfrom the group consisting of (C₁-C₄)alkyl, halo, (C₁-C₄)alkoxy, aminoand nitro;

A² is deleted or D- or L- of the following amino acids: Ile, Val, Leu,Nle, Anb, Aib, Pro, Gln or Asn;

A³ is deleted or D- or L- of the following amino acids: Asn, Gln, Glu,Asp, Orn, Lys, Dpr or Cys;

A⁴ is deleted or D- or L- of the following amino acids: Ile, Val, Leu,Nle, Anb, Aib or Pro;

A⁵ is deleted or D- or L- of the following amino acids: Ile, Val, Leu,Nle, Anb, Aib, Pro, Glu, Asp, Orn, Lys, Dpr or Cys;

A⁶ is deleted or D- or L- of the following amino acids: Thr, Ser, Trp,Tyr, Fla, Bth, Nal, Tic, Tic-OH, Dip, Bip or optionally substituted Phewhere the Phe is optionally substituted with one to five substituentsselected from the group consisting of (C₁-C₄)alkyl, halo, (C₁-C₄)alkoxy,amino and nitro;

A⁷ is deleted or D- or L- of the following amino acids: Arg, Lys,homo-Arg, dialkyl-homo-Arg, Lys-ε-NH-R⁷ or Orn;

A⁸ is deleted or D- or L- of the following amino acids: Nva, Val, Ile,Leu, Nle, Anb, Aib, Pro, Gln, Asn, Glu, Asp, Orn, Lys, Dpr or Cys;

A⁹ is deleted or D- or L- of the following amino acids: Arg, Lys,homo-Arg, dialkyl-homo-Arg, Lys-ε-NH-R⁷ or Orn; and

A¹⁰ is deleted or D- or L- of the following amino acids: Tyr, Trp, Fla,Bth, Nal, Tic, Tic-OH, Dip, Bip, tyramine or optionally substituted Phewhere the Phe is optionally substituted with one to five substituentsselected from the group consisting of (C₁-C₄)alkyl, halo, (C₁-C₄)alkoxy,amino and nitro, or the corresponding decarboxylated optionallysubstituted Phe;

where R⁷ for each occurrence is independently selected from the groupconsisting of H.sub.₁ (C₁-C₁₀)alkyl and (C₆-C₁₈) aryl, provided that notall of A₁ to A₁₀ are deleted at the same time. See U.S. Pat. No.6,046,167.

A particular group of compounds of the immediately foregoing group ofcompounds is

or a pharmaceutically acceptable salt thereof.

PYY and PYY agonists may be produced by recombinant DNA technology bychemical synthesis, from natural sources, or by any combination thereof.PYY and PYY agonists may be modified by well known processes such asamidation, glycosylation, acylation (e.g. acetylation), sulfation,phosphylation, cyclization, lipidization and pegylation. Methods forlipidization with fatty acid derivatives of sulfhydryl-containingcompounds are disclosed in U.S. Pat. No. 5,936,092; U.S. Pat. No.6,093,692; and U.S. Pat. No. 6,225,445. Fatty acid derivatives ofsulfhydryl-containing PYY and PYY agonists comprising fattyacid-conjugated products with a disulfide linkage are employed fordelivery of the PYY and PYY agonists to neuronal cells and tissues. Thismodification markedly increases the absorption of the compounds relativeto the rate of absorption of the unconjugated compounds, as well asprolonging blood and tissue retention of the compounds. Moreover, thedisulfide linkage in the conjugate is quite labile in the cells and thusfacilitates intracellular release of the intact compounds from the fattyacid moieties.

Fatty acids, as constituents of phospholipids, make up the bulk of cellmembranes. Due to their lipidic nature, fatty acids can easily partitioninto and interact with the cell membrane in a non-toxic way. Therefore,fatty acids represent potentially a useful carrier ligand for thedelivery of proteins and peptides. Strategies that may use fatty acidsin the delivery of proteins and peptides include the covalentmodification of proteins and peptides and the use of fatty acidemulsions.

To prepare such conjugates, a sulfhydryl-containing PYY and PYY agonistis attached to a fatty acid derivative via a reversible, biodegradabledisulfide bond. Such a conjugate is expected to bind to the apical sideof a cell membrane, reach the basolateral membrane of the GI-epitheliumas a result of membrane transport and turnover, and become released intointerstitial fluid as the result of disulfide bond reduction.

Such lipidized PYY and PYY agonist compounds have the general formula

in which P is a residue derived from a PYY or PYY agonist; R¹ ishydrogen, lower alkyl or aryl; R² is a lipid-containing moiety and R³ is—OH, a lipid-containing moiety or an amino acid chain comprising one or2 amino acids and terminating in —CO₂H or —COR². See U.S. Pat. No.5,936,092. These conjugates are particularly useful for increasing theabsorption and prolonging blood and tissue retention of PYY and PYYagonists.

Typical alkyl groups include C₁₋₆ alkyl groups including methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,2-pentyl, 3-pentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl,2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, and the like.

Preferred aryl groups are C₆₋₁₄ aryl groups and typically includephenyl, naphthyl, fluorenyl, phenanthryl, and anthracyl groups.

The term “lipid-containing moiety” refers to either a lipid group per seor a hydrocarbon-based group (in particular, one or more amino acids)comprising a lipid group. By the term “lipid group” is meant ahydrophobic substituent consisting of 4 to 26 carbon atoms, preferably 5to 19 carbon atoms. Suitable lipid groups include, but are not limitedto, the following: palmityl (C₁₅H₃₁,), oleyl (C₁₅H₂₉), stearyl (C₁₇H₃₅),cholate; and deoxycholate.

PCT Application No. WO 00/34236 describes drug-carrier conjugates andsynthetic strategies for their production, as well as synthetic methods,intermediates, and final products useful for the uptake and release ofbiologically-active amino group containing compounds. Such lipidized PYYand PYY agonist compounds have general Formula I

in which R² is selected from the group consisting of hydrogen, halo,alkyl, or aryl, wherein the alkyl or aryl groups are optionallysubstituted with one or more alkoxy, alkoxyalkyl, alkanoyl, nitro,cycloalkyl, alkenyl, alkynyl, alkanoyloxy, alkyl or halogen atoms;R³ is a lipophilic group; one of R⁴ and R⁵ is a PYY or a PYY agonist andthe other of R⁴ and R⁵ is OR⁶ where R⁶ is hydrogen, an alkali metal or anegative charge;X is oxygen or sulfur;Y is a bridging natural or unnatural amino acid; n is zero or 1; and mis an integer from zero to 10.

Typical alkyl groups include C₁₋₆ alkyl groups including methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,2-pentyl, 3-pentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl,2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, and the like.

Typical alkoxy groups include oxygen substituted by any of the alkylgroups mentioned above.

Typical alkoxyalkyl groups include any of the above alkyl groupssubstituted by an alkoxy group, such as methoxymethyl, ethoxymethyl,propoxymethyl, butoxymethyl, pentoxymethyl, hexoxymethyl, methoxyethyl,methoxypropyl, methoxybutyl, methoxypentyl, methoxyhexyl, and the like.

Preferred aryl groups are C₆₋₁₄ aryl groups and typically includephenyl, naphthyl, fluorenyl, phenanthryl, and anthracyl groups.

Typical alkoxy substituted aryl groups include the above aryl groupssubstituted by one or more of the above alkoxy groups, e.g.,3-methoxyphenyl, 2-ethoxyphenyl, and the like.

Typical alkyl substituted aryl groups include any of the above arylgroups substituted by any of the C₁₋₆ alkyl groups, including the groupPh(CH₂)n, where n is 1-6, for example, tolyl, o-, m-, and p-xylyl,ethylphenyl, 1-propylphenyl, 2-propylphenyl, 1-butylphenyl,2-butylphenyl, t-butylphenyl, 1-pentylphenyl, 2-pentylphenyl,3-pentylphenyl.

Typical alkenyl groups include C₂₋₆ alkenyl groups, e.g. ethenyl,2-propenyl, isopropenyl, 2-butenyl, 3-butenyl, 4-pentenyl, 3-pentenyl,2-pentenyl, 5-hexenyl, 4-hexenyl, 3-hexenyl, and 2-hexenyl groups.

Typical alkynyl groups include C₂₋₆ alkynyl groups e.g. enthynyl,2-propenyl, 2-butynyl, 3-butynyl, 4-pentynyl, 3-pentynyl, 2-pentynyl,5-hexynyl, 4hexynyl, 3-hexynyl, and 2-hexynyl groups.

Typical alkenyl or alkynyl substituted aryl groups include any of theabove C₆₋₁₄ arylgroups substituted by any of the above C₂₋₆ alkenyl orC₂₋₆ alkynyl groups, e.g., ethenylphenyl, 1-propenylphenyl,2-propenylphenyl, 1butenylphenyl, 2-butenylphenyl, 1-pentenylphenyl,2-pentenylphenyl, 3-pentenylphenyl, 1-hexenylphenyl, 2-hexenylphenyl,3-hexenylphenyl, ethynylphenyl, 1-propynylphenyl, 2-propynylphenyl,1-butynylphenyl, 2-butynylphenyl, 1-pentynylphenyl, 2-pentynylphenyl,3-pentynylphenyl, 1-hexynylphenyl, 2-hexynylphenyl, 3-hexynylphenylgroups.

Typical halo groups include fluorine, chlorine, bromine, and iodine.

Typical halo substituted alkyl groups include C₁₋₆ alkyl groupssubstituted by one or more fluorine, chlorine, bromine, or iodine atoms,e.g., fluoromethyl, difluoromethyl, trifluoromethyl, pentafluoroethyl,1,1-difluoroethyl, and trichloromethyl groups.

Typical alkanoyl groups include C₁₋₅C(═O)-alkanoyl groups, e.g., acetyl,propionyl, butanoyl, pentanoyl, and hexanoyl groups, or by anarylalkanoyl group, e.g., a C₁₋₅C(═O)-alkanoyl group substituted by anyof the above aryl groups.

Typical cycloalkyl groups include C₃₋₈ cycloalkyl groups includingcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl groups.

The term “lipophilic group” as used herein refers to either a naturallyoccurring lipid per se, a hydrophobic branched or unbranched hydrocarboncomprising about 4 to about 26 carbon atoms, preferably about 5 to about19 carbon atoms, a fatty acid or ester thereof, or a surfactant.Suitable lipophilic groups include, but are not limited to, long chainalkanoyl groups including: palmityl (C₁₅H₃₁), oleyl (C₁₅H₂₉), stearyl(C₁₇H₃₅), lauryl (C₁₁H₂₃), cholyl, and myristyl (C₁₃H₂₇)

The Term “Natural or Unnatural Amino Acid” as Used Herein Refers to anyof the 21 naturally occurring amino acids as well as D-form amino acids,blocked L- and D-form amino acids such as those blocked by amidation oracylation, substituted amino acids (e.g., those substituted with asterically hindered alkyl group or a cycloalkyl group such ascyclopropyl or cyclobutyl) in which the substitution introduces aconformational restraint in the amino acid. The preferred naturallyoccurring amino acids for use in the present disclosure as amino acidsor components of a peptide or protein are alanine, arginine, asparagine,aspartic acid, citrulline, cysteine, cystine, y-glutamic acid,glutamine, glycine, histidine, isoleucine, norleucine, leucine, lysine,methionine, ornithine, phenylalanine, proline, hydroxyproline, serine,threonine, tryptophan, tyrosine, valine, γ-carboxyglutamate, orO-phosphoserine. The preferred non-naturally occurring amino acids foruse in the present disclosure as amino acids or components of peptidesor proteins are any of the β-amino acids, e.g., α-alanine, γ-aminobutyric acid, γ-amino butyric acid, γ-(aminophenyl)butyric acid, α-aminoisobutyric acid, ε-amino caproic acid, 7-amino heptanoic acid, aminobenzoic acid, aminophenyl acetic acid, aminophenyl butyric acid,cysteine (ACM), methionine sulfone, phenylglycine, norvaline, ornithine,δ-ornithine, ρnitro-phenylalanine,1,2,3,4-terahydroisoquinoline-3-carboxylic acid and thioproline.

The present disclosure is also directed to methods of preparinglipidized conjugates of PYY and PYY agonists, pharmaceuticalcompositions comprising lipidized conjugates of PYY and PYY agonists,and methods of increasing the delivery of amino group-containing PYY andPYY agonists into a cell.

Also provided by the disclosure are chemically modified derivatives ofPYY and PYY agonists which may provide additional advantages such asincreased solubility, stability and circulating time of the polypeptide,or decreased immunogenicity (see U.S. Pat. No. 4,179,337). Such modifiedderivatives include PYY and PYY agonists modified by pegylation. Theterms “pegylated” and “pegylation” refer to the process of reacting apoly(alkylene glycol), preferably an activated poly(alkylene glycol),with a facilitator such as an amino acid, e.g. lysine, to form acovalent bond. Although “pegylation” is often carried out usingpoly(ethylene glycol) or derivatives thereof, such as methoxypoly(ethylene glycol), the term is not intended to be so limited here,but is intended to include any other useful poly(alkylene glycol), suchas, for example poly(propylene glycol).

The chemical moieties for derivitization may also be selected from watersoluble polymers such as polyethylene glycol, ethylene glycol/propyleneglycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcoholand the like. The polypeptides may be modified at random positionswithin the molecule, or at predetermined positions within the moleculeand may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72,1996; Vorobjev et al., Nucleosides Nucleotides 18:2745-2750, 1999; andCaliceti et al., Bioconjug. Chem. 10:638-646, 1999.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the polypeptides or proteins with consideration of effectson functional or antigenic domains of the polypeptides or proteins.There are a number of attachment methods available to those skilled inthe art, e.g., EP 0 401 384 (coupling PEG to G-CSF), see also Malik etal., Exp. Hematol. 20:1028-1035, 1992 (reporting pegylation of GM-CSFusing tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins andpolypeptides via linkage to any of a number of amino acid residues. Forexample, polyethylene glycol can be linked to proteins and polypeptidesvia covalent bonds to lysine, histidine, aspartic acid, glutamic acid,or cysteine residues. One or more reaction chemistries may be employedto attach polyethylene glycol to specific amino acid residues (e.g.,lysine, histidine, aspartic acid, glutamic acid, or cysteine) of thepolypeptide or protein or to more than one type of amino acid residue(e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine andcombinations thereof) of the protein or polypeptide.

One may specifically desire proteins and polypeptides chemicallymodified at the N-terminus. Using polyethylene glycol as anillustration, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins and polypeptides may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein or polypeptide either directly or by anintervening linker. Linkerless systems for attaching polyethylene glycolto proteins and polypeptides are described in Delgado et al., Crit. Rev.Thera. Drug Carrier Sys. 9:249-304, 1992; Francis et al., Intern. J. ofHematol. 68:1-18, 1998; U.S. Pat. No. 4,002,531; U.S. Pat. No.5,349,052; WO 95/06058; and WO 98/32466.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins and polypeptides without an intervening linkeremploys tresylated MPEG, which is produced by the modification ofmonmethoxy polyethylene glycol (MPEG) using tresylchloride(ClSO₂CH₂CF₃). Upon reaction of the protein or polypeptide withtresylated MPEG, polyethylene glycol is directly attached to aminegroups of the protein or polypeptide. Thus, the disclosure includesprotein-polyethylene glycol conjugates produced by reacting proteins andpolypeptides with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins and polypeptidesusing a number of different intervening linkers. For example, U.S. Pat.No. 5,612,460 discloses urethane linkers for connecting polyethyleneglycol to proteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein or polypeptide by alinker can also be produced by reaction of proteins or polypeptides withcompounds such as MPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-ρ-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber of additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins andpolypeptides are described in WO 98/32466.

The number of polyethylene glycol moieties attached to each protein orpolypeptide (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins and polypeptides may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein or polypeptide molecule.Methods for determining the degree of substitution are discussed, forexample, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304, 1992.

The proteins and polypeptides containing substantially non-antigenicpolymers, preferably poly(alkylene glycols) may be prepared, forexample, as described in U.S. Pat. No. 5,428,128; U.S. Pat. No.6,127,355; and U.S. Pat. No. 5,880,131.

To effect covalent attachment of poly(ethylene glycol) (PEG) to aprotein or polypeptide, the hydroxyl end groups of the PEG must first beconverted into reactive functional groups. This process is frequentlyreferred to as “activation” and the product is called “activated PEG.”Methoxy poly(ethylene glycol) (mPEG), distally capped with a reactivefunctional group is often used. One such activated PEG is succinimidylsuccinate derivative of PEG (SS-PEG), See also Abuchowski et al., CancerBiochem. Biophys. 7:175-186, 1984; and U.S. Pat. No. 5,122,614 whichdiscloses poly(ethylene glycol)-N-succinimide carbonate and itspreparation.

Alternative substantially non-antigenic polymers that may be employed inthe practice of the present disclosure include materials such asdextran, polyvinyl pyrrolidones, polysaccharides, starches, polyvinylalcohols, polyacrylamides, or other similar non-immunogenic polymers.Those of ordinary skill in the art will realize that the foregoing aremerely illustrative and not intended to restrict the type of polymericsubstances suitable for use herein.

In one aspect of the disclosure, the polymer is introduced into thepeptide or protein molecule after being functionalized or activated forreaction and attachment to one or more amino acids. By activation, it isunderstood by those of ordinary skill in the art that the polymer isfunctionalized to include a desired reactive group. See, for example,U.S. Pat. No. 4,179,337 and U.S. Pat. No. 5,122,614. In this embodiment,the hydroxyl end groups of poly(alkylene glycols) are converted andactivated into reactive functional groups.

In another aspect of the disclosure, the polymer is conjugated to afacilitator moiety prior to being introduced into the polypeptide orprotein molecule. The facilitator moiety is preferably an amino acidsuch as lysine, however, non-amino acid moieties are also contemplated.Within the aspect, there are included multifunctionalized organicmoieties such as alkyls or substituted alkyls. Such moieties can beprepared to have a nucleophilic functional group such as an amine and anelectrophilic group such as an acid as well as a suitably functionalizedregion for conjugating with the desired polymer or polymers.

The facilitator moieties allow easier inclusion of a polymer into thepeptide or protein molecule during synthesis. For example, poly(alkyleneglycols) coupled to facilitator amino acids or amino acid residues inpolypeptides or proteins by means of suitable coupling agents areillustrative. A useful review of a number of coupling agents known inthe art appears in Dreborg et al., Critical Reviews in Therapeutic DrugCarrier Systems 6(4):315-165, 1990, see especially, pp. 317-320.

Pegylated PYY peptides and agonists can also be of the general formula

wherein:

D is a residue of a PYY peptide or agonist;

X is an electron withdrawing group;

Y and Y′ are independently O or S;

(n) is zero (0) or a positive integer, preferably from 1 to about 12;

R₁ and R₂ are independently selected from the group consisting of H,C₁₋₆ alkyls, aryls, substituted aryls, aralkyls, heteroalkyls,substituted heteroalkyls, and substituted C₁₋₆ alkyls;

R₃ is a substantially non-antigenic polymer, C₁₋₁₂ straight or branchedalkyl or substituted alkyl, C₅₋₈ cycloalkyl or substituted cycloalkyl,carboxyalkyl, carboalkoxy alkyl, dialkylaminoalkyl, phenylalkyl,phenylaryl or

and

R₄ and R₅ are independently selected from the group consisting of H,C₁₋₆ alkyls, aryls, substituted aryls, aralkyls, heteroalkyls,substituted heteroalkyls and substituted C₁₋₆ alkyls or jointly form acyclic C₅-C₇ ring. See U.S. Pat. No. 6,127,355.

Typical alkyl groups include C₁₋₆ alkyl groups including methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,2-pentyl, 3-pentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl,2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, and the like.

Preferred aryl groups are C₆₋₁₄ aryl groups and typically includephenyl, naphthyl, fluorenyl, phenanthryl, and anthracyl groups.

Typical alkyl substituted aryl groups include any of the above arylgroups substituted by any of the C₁₋₆ alkyl groups, including the groupPh(CH₂)n, where n is 1-6, for example, tolyl, o-, m-, and p-xylyl,ethylphenyl, 1-propylphenyl, 2-propylphenyl, 1-butylphenyl,2-butylphenyl, t-butylphenyl, 1-pentylphenyl, 2-pentylphenyl,3-pentylphenyl.

Typical cycloalkyl groups include C₃₋₈ cycloalkyl groups includingcyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl andcyclooctyl groups.

Typical electron withdrawing groups include O, NR₁, S, SO and SO₂,wherein R₁ is defined above.

GLP-1 and Agonists Thereof

GLP-1 is produced from preproglucogon, which is a 160 amino acidpolypeptide, in the central nervous system (CNS) and the intestine. Itis released into the circulation in response to nutrient intake.Physiological actions of GLP-1 in man include stimulation of insulinrelease, suppression of gastric acid secretion and slowing of gastricemptying.

GLP-1 (1-37) (SEQ ID NO: 336) is the initial product of the processingof preproglucagon. GLP-1 (1-37) is amidated by post-translationalprocessing to yield GLP-1 (1-36) NH (SEQ ID NO 337), or is enzymaticallyprocessed to give GLP-1 (7-37) (SEQ ID NO: 338). GLP-1 (7-37) can beamidated to give GLP-1 (7-36) amide (SEQ ID NO: 339). The sequences ofhuman GLP-1 are given below:

GLP-1 (1-37): (SEQ ID NO: 336)His Asp Glu Phe Glu Arg His Ala Glu Gly Thr PheThe Ser Asp Val Ser Ser Tyr Leu Glu Gly Gly AlaAla Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly. GLP-1 (1-36) amide:(SEQ ID NO: 337) His Asp Glu Phe Glu Arg His Ala Glu Gly Thr PheThe Ser Asp Val Ser Ser Tyr Leu Glu Gly Gly AlaAla Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg NH₂, GLP-1 (7-37):(SEQ ID NO: 338) His Ala Glu Gly Thr Phe The Ser Asp Val Ser SerTyr Leu Glu Gly Gly Ala Ala Lys Glu Phe Ile AlaTrp Leu Val Lys Gly Arg Gly. GLP-1 (7-36) amide: (SEQ ID NO: 339)His Ala Glu Gly Thr Phe The Ser Asp Val Ser SerTyr Leu Glu Gly Gly Ala Ala Lys Glu Phe Ile AlaTrp Leu Val Lys Gly Arg NH₂.

A GLP-1 agonist is a peptide, small molecule, or chemical compound thatpreferentially binds to the GLP-1 receptor and stimulates the samebiological activity as does GLP-1. In one embodiment, an agonist for theGLP-1 receptor binds to the receptor with an equal or greater affinitythan GLP-1. In another embodiment, an agonist selectively binds theGLP-1 receptor, as compared to binding to another receptor. Exendin-4,which is a 39-amino acid peptide isolated from the salivary glands ofthe Gila monster (Heloderma suspectum) (Eng J et al J Biol Chem267:7402-7405, 1992) is an example of an agonist at the GLP-1 receptor.Molecules derived from exendin-4 and that also have GLP-1 agonistactivity are further examples of GLP-1 agonists. GLP-1 agonists includeGLP-1 related peptides and peptides that result from natural orsynthetic enzymatic or chemical processing of preproglucagon or of aGLP-1 peptide or a related peptide.

Any compound that is described as being a GLP-1 agonist may be used inthe present invention, as may any compound that is tested for GLP-1agonist activity, for example, as described above, and found to functionas a GLP-1 agonist. A recombinant GLP-1 receptor suitable for use inscreening is disclosed in WO93/19175. Many GLP-1 agonists are known andare described in the art. Examples of published patent specificationsthat disclose GLP-1 agonists are the following: WO2002/67918,WO2002/66479, WO2002/03978, WO2001/89554, WO2001/14386, WO2001/66135,WO2001/35988, WO2001/14368, WO2001/04156, WO2000/78333, WO2000/59887,WO2000/42026, EP 0955314, and WO99/43707. Examples of GLP-1 agonists areArg34, Lys26(N-epsilon-(gamma-Glu(N-alpha-hexadecanoyl)))-GLP-1 (7-37),IP7-GLP-1 (7-37)OH.

GLP-1 or an agonist thereof may be administered according to the presentinvention peripherally at a dose of, for example, 0.1 nmoles or more perkg body weight of the subject, for example, 0.2 nmoles or more, forexample, 0.4 nmoles or more, for example, 0.6 nmoles or more, forexample, 0.8 nmoles or more, for example, 1.0 nmole or more, forexample, 1.2 nmoles or more, for example, 1.4 nmoles or more, forexample, 1.6 nmoles or more, for example, 1.8 nmoles or more, forexample, 2.0 nmoles or more, for example, 2.2 nmoles or more, forexample, 2.4 nmoles or more, for example, 2.6 nmoles or more, forexample, 2.8 nmoles, for example, 3.0 nmoles or more, for example, up to3.2 nmoles per kg body weight. The amount used may be up to 3.0 nmolesper kg body weight, for example, up to 2.8 nmoles, for example, up to2.6 nmoles, for example, up to 2.4 nmoles, for example, up to 2.2nmoles, for example, up to 2.0 nmoles, for example, up to 1.8 nmoles,for example, up to 1.4 nmoles, for example, up to 1.2 nmoles, forexample, up to 1.0 nmoles, for example, up to 0.8 nmoles, for example,up to 0.6 nmoles, for example, up to 0.4 nmoles, for example, up to 0.2nmoles per kg body weight. The dose is generally in the range of from0.1 to 3.2 nmoles per kg body weight, for example, within anycombination of upper and lower ranges given above.

We have shown that intracerebroventricular (ICV) administration of GLP-1to rats potently inhibits food intake. This effect is blocked by thespecific GLP-1 receptor antagonist, exendin 9-39. GLP-1 receptors arefound in the brainstem, arcuate nucleus and PVN. Following ICV GLP-1administration, c-fos is expressed in the arcuate nucleus and PVN.However, peripheral administration of GLP-1 in man and rat also inhibitsfood intake and results in c-fos expression in the brainstem of rats. Asis disclosed herein, the effect on 1-hour food intake in non-fasted ratsof IP administration of GLP-1 is not influenced by the presence ofconcomitant exendin 9-39, a GLP-1 receptor agonist, in the arcuatenucleus of the rat. This indicates that circulating GLP-1 acts via thebrainstem rather than the arcuate nucleus.

These and other findings suggest that the main site for appetiteinhibition by peripheral GLP-1 is the dorsal vagal complex, acting inpart directly through the area postrema. However, the physiologicalimportance of this in man is currently unclear.

As disclosed herein, when administered to humans, PYY was found toreduce appetite. When infused into humans at physiological post-prandiallevels, PYY₃₋₃₆ significantly decreased appetite and reduced food intakeby a third over 12 hours, and even by a third over 24 hours. Both theeffect itself and the duration of the effect are surprising andunpredictable, as they occurred for many hours after the hormone hadbeen cleared from the circulation. The effects, which are produced atphysiological levels of the peptide, are strong indications that PYYacts in vivo to regulate feeding behavior.

As disclosed herein, peripheral administration of PYY₃₋₃₆ in the ratcaused an increase of c-fos immunoreactivity in the arcuate nucleus ofthe hypothalamus and a decrease in hypothalamic neuropeptide Y (NPY)mRNA. Further, electro-physiological studies demonstrated that PYY₃₋₃₆inhibits synaptic activity of the NPY nerve terminals and thus activatesPOMC neurons, which are known to receive inhibitory NPY synaptic inputs.

Without being bound by theory, these results demonstrate that the guthormone PYY₃₋₃₆ can act via the neuropeptide Y Y2 receptor. Thishypothesis is supported by the observation that when PYY₃₋₃₆ wasadministered to neuropeptide Y Y2 receptor null mice (Y2R gene knock outmice), no inhibition of feeding was observed. Administration of PYY₃₋₃₆to wild type littermates of the Y2R null mice was fully effective ininhibiting feeding.

Thus, a novel gut-brain pathway that inhibits feeding after meals isdescribed. Without being bound by theory, the natural pathway involvesrelease of PYY from the gut, its conversion to PYY₃₋₃₆, which acts as anagonist on the neuropeptide Y Y2 receptor (NPY Y2 receptor) in thebrain. The NPY Y2 receptor acts as a inhibitory pre-synaptic receptorreducing release of neuropeptide Y, which is a most potent stimulator offeeding, and also acting on the anorexigenic melanocortin systems, theresult of the NPY Y2 receptor activity being to suppress appetite anddecrease food intake. The action of PYY₃₋₃₆ may occur in the arcuatenucleus of the hypothalamus, but other areas may be also be involved.

The results obtained show that PYY₃₋₃₆, a gut hormone that circulates inthe blood, inhibits appetite at physiological concentrations, and thatthe inhibitory effect is observed even for some hours after the hormonehas been cleared from the blood. This effect has been observed in allspecies tested, i.e. in mouse, rat and human. The circulating guthormone appears to act via hypothalamic circuits. The reduction ofmessenger RNA, necessary for the synthesis of brain appetite regulatinghormones, in particular of hypothalamic NPY mRNA may be a possiblemechanism for the long action of PYY₃₋₃₆.

When PYY₃₋₃₆ was co-administered parenterally, either IP or IV withGLP-1, a clear synergistic reduction in food intake was produced. Thiscontrasts with the results obtained with oxyntomodulin, where there wasno synergistic effect, only a sum of the individual effects. Asindicated above, PYY₃₋₃₆ is considered to act via the arcuate nucleus.Oxynotmodulin is also considered to act via that region of the brain.GLP-1, however, is believed to act via the brainstem, as explainedabove. While not being limited to the following, it appears that, whenagents that act via different areas of the brain and/or by differentneurological routes are administered parenterally, they produce asynergistic affect, whereas when they act by the same area and/or routethey do not.

These results indicate the importance in vivo of the interaction of guthormones on the regulation of appetite and food intake. The presentinvention demonstrates that, surprisingly, it is possible to regulateappetite and food intake more effectively by informed choice of agents.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Material and Methods

Generation of POMC-EGFP mice: The EGFP cassette contains its own Kozakconsensus translation initiation site along with SV40 polyadenylationsignals downstream of the EGFP coding sequences directing properprocessing of the 3′ end of the EGFP mRNA. The EGFP cassette wasintroduced by standard techniques into the 5′ untranslated region ofexon 2 of a mouse Pomc genomic clone containing 13 kb of 5′ and 2 kb of3′ flanking sequences (Young et al., J Neurosci 18, 6631-40, 1998). Thetransgene was microinjected into pronuclei of one-cell stage embryos ofC57BL/6J mice (Jackson Laboratories) as described (Young et al., JNeurosci 18, 6631-40, 1998). One founder was generated and bred towildtype C57BL/6J to produce N₁ hemizygous mice. In addition, N₂ andsubsequent generations of mice homozygous for the transgene were alsogenerated. The mice are fertile and have normal growth and development.

Immunofluorescence and GFP co-localization: Anesthetized mice wereperfused transcardially with 4% paraformaldehyde and free-floating brainsections prepared with a vibratome. Sections were processed forimmunofluorescence and colocalization of GFP fluorescence using standardtechniques. Primary antisera and their final dilutions were rabbitanti-β-endorphin, 1:2500 v/v; rabbit anti-NPY, 1:25,000 v/v (AlanexCorp.); rabbit anti-ACTH, 1:2000 v/v; and mouse anti-TH, 1:1000 v/v(Incstar). After rinsing, sections were incubated with 10 mg/mlbiotinylated horse anti-mouse/rabbit IgG (Vector Laboratories) followedby Cy-3 conjugated streptavidin, 1:500 v/v (Jackson ImmunoresearchLaboratories). Photomicrographs were taken on a Zeiss Axioscop usingFITC and RITC filter sets (Chroma Technology Corp.).

Electrophysiology (Example 2): 200 μm thick coronal slices were cut fromthe ARC of four-week old male POMC-EGFP mice. Slices were maintained in(in mM) [NaCl, 126; KCl, 2.5; MgCl₂, 1.2; CaCl₂.2H₂O, 2.4; NaH₂PO₄.H₂O,1.2; NaHCO₃, 21.4; Glucose, 11.1] (Krebs) at 35° C. and saturated with95% O₂ 5% CO₂ for 1 hour(hr) prior to recordings. Recordings were madein Krebs at 35° C. Slices were visualized on an Axioskop FS2 (Zeiss)through standard infra red optics and using epifluoresence through aFITC filter set (see FIG. 1 c). Whole cell recordings were made fromfluorescent neurons using an Axopatch 1D amplifier (Axon Instruments)and Clampex 7 (Axon Instruments). Resting membrane potentials weredetermined using an event detection protocol on a PowerLab system (ADInstruments, Mountain View, Calif.) to average expanded traces of themembrane potential. Drugs were applied to the bath over the timesindicated. The resting membrane potential was stable for up to an hourin cells treated with Krebs alone. I-V relationships for the Met-Enkcurrents were established using a step protocol; (−60 mV holdingpotential, sequentially pulsed (40 ms) from −120 to −50 mV, cells werereturned to −60 mV for 2 s between voltage steps). The protocol wasrepeated after Met Enk addition. The net current was the differencebetween the two I-V relationships. This protocol was repeated in Krebswith 6.5 mM K⁺. I-V relationships to identify the postsynaptic leptincurrent were performed similarly with slow voltage ramps (5 mV/s from−100 to −20 mV) before and 10 minutes after the addition of leptin (100nM). GABAergic IPSCs were recorded using a CsCl internal electrodesolution (in mM) [CsCl, 140; Hepes, 10; MgCl₂, 5; Bapta, 1; (Mg)-ATP, 5;(Na)GTP, 0.3]. Both mini IPSCs and large amplitude (presumablymultisynaptic) IPSCs were observed in the untreated slices. TTX (1 μM)abolished large IPSCs. Data were acquired before and after addition ofdrug for the times indicated on the figures at a −50 mV holdingpotential in 2 s. sweeps every 4 s. Mini postsynaptic currents wereanalyzed using Axograph 4 (Axon Instruments). IPSCs and excitatorypostsynaptic currents (EPSCs) were distinguished on the basis of theirdecay constants; additionally picrotoxin (100 μM) blocked all IPSCs.POMC neurons receive a low EPSC tone and the frequency was not modulatedby any of the treatments described here.

Immunostaining for light and electron microscopy: Doubleimmunocytochemistry for NPY and POMC using different colordiaminobenzidine(DAB) chromogens was carried out on fixed mousehypothalami according to published protocols (Horvath et al.,Neuroscience 51, 391-9, 1992). For electron microscopy, preembeddingimmunostaining for β-endorphin was using an ABC Elite kit (VectorLaboratories) and a DAB reaction followed by post-embedding labeling ofGABA and NPY using rabbit anti-GABA, 1:1000 v/v and gold conjugated (10nm) goat anti-rabbit IgG or sheep anti-NPY and gold conjugated (25 nm)goat anti-sheep IgG. Finally, sections were contrasted with saturateduranyl acetate (10 minutes) and lead citrate (20-30 s) and examinedusing a Philips CM-10 electron microscope.

Animals: Male Wistar rats (200-2500, 7-8 weeks old (Charles RiverLaboratories, United Kingdom) were maintained under controlledtemperature (21-23° C.) and light conditions (lights on 07:00-19:00)with ad libitum access to water and food (RM1 diet; SDS Ltd., Witham,United Kingdom) except where stated. Arcuate and paraventricular nucleicannulations and injections were performed as previously described(Glaum et al., Mol. Pharmacol. 50, 230-5, 1996; Lee et al., J. Physiol(Lond) 515, 439-52; 1999; Shiraishi et al., Nutrition 15, 576-9, 1999).Correct intranuclear cannula placement was confirmed histologically atthe end of each study period (Glaum et al., Mol. Pharmacol. 50, 230-5,1996; Lee et al., J. Physiol (Lond) 515, 439-52, 1999; Shiraishi et al.,Nutrition 15, 576-9, 1999). All animal procedures were approved underthe British Home Office Animals (Scientific Procedures) Act, 1986. Allinjection studies on fasting animals were performed in the earlylight-phase (0800-0900). All dark-phase feeding studies injections wereperformed just prior to lights off.

Male Pomc-EGFP mice were studied at 5-6 weeks of age and were generatedas described above. Y2r-null mice were generated using Cre-lox Pmediated recombination, which results in the germline deletion of theentire coding region of the Y2 receptor. All Y2r-null mice weremaintained on a mixed C57/B16-129SvJ background. Male mice aged 8-12weeks and between 20-30 g bodyweight were kept under controlledtemperature (21-23° C.) and light conditions (lights on 06:00-18:00)with ad libitum access to water and food (Gordon's Speciality Stockfeeds) except where stated. All studies were performed in the earlylight-phase (0700-0800).

Intraperitoneal injections: Rats were accustomed to IP injection byinjections of 0.5 ml saline on the two days prior to study. For allstudies, animals received an IP injection of PYY₃₋₃₆, GLP-1 or saline in500 μl (for rats) or 100 μl (for mice).

Electrophysiology: Whole cell patch clamp recordings were made from POMCneurons in the hypothalamus of 180 μm thick coronal slices fromPomc-EGFP mice, as previously reported (Cowley et al., Nature 411,480-484, 2001). “Loose cell-attached” recordings were made usingextracellular buffer in the electrode solution, and maintaining sealresistance between 3-5 Mohm throughout the recording. Firing rates wereanalysed using mini-analysis protocols (MiniAnalysis, Jaejin Software,NJ). Vehicle controls were used in this system, previously validated forthe electrophysiological actions of neuropeptides (Cowley et al., Nature411, 480-484, 2001). Data were analysed by ANOVA, Neuman-Keuls posthoccomparison, and Wilcoxon Signed Rank Test.

Hypothalamic explants: Male Wistar rats were killed by decapitation andthe whole brain immediately removed, mounted with the ventral surfaceuppermost and placed in a vibrating microtome (Biorad, MicrofieldScientific Ltd., Devon, UK). A 1.7 mm slice was taken from the base ofthe brain to include the PVN and the ARC and immediately transferred to1 ml of artificial CSF (aCSF) (Kim et al., J. Clin. Invest. 105,1005-11, 2000) equilibrated with 95% O₂ and 5% CO₂ and maintained at 37°C. After an initial 2-hour equilibration period, with aCSF replacedevery 60 minutes, the hypothalami were then incubated for 45 minutes in600 μl aCSF (basal period) before being exposed to the Y2A (50 nM) in600 μl aCSF. Finally, the viability of the tissue was verified by a 45minute exposure to 56 mM KCL; isotonicity was maintained by substitutingK⁺ for Na⁺. At the end of each period, the aCSF was removed and frozenat −20° C. until assayed for NPY and αMSH by radioimmunoassay.

C-fos expression: C-fos expression was measured in adult Wistar rats andPomc-EGFP mice 2 hours after IP administration of saline or PYY₃₋₃₆ (5μg/100 g) using standard immunohistochemical techniques (Hoffman et al.,Front. Neuroendocrinol. 14, 173-213, 1993). Data were obtained from 3rats and 5 mice in each group. For the Pomc-EGFP mice 5 anatomicallymatched arcuate nucleus sections (Franklin et al., The Mouse Brain inStereotaxic Coordinates, Academic Press, San Diego, 1997) were countedfrom each animal, and images acquired using a Leica TSC confocalmicroscope (Grove et al., Neuroscience 100, 731-40, 2000).

RNase protection assay (RPA): Total RNA was extracted from hypothalami(Trizol, Gibco). RPAs were performed (RPAIII kit, Ambion) using 5 μg RNAand probes specific for NPY, αMSH and β actin (internal standard). Foreach neuropeptide, the ratio of the optical density of the neuropeptidemRNA band to that of β actin was calculated. Neuropeptide mRNAexpression levels are expressed relative to saline control (mean±s.e.m.n=4 per group). The statistical analysis used was ANOVA, with Bonferronipost hoc analysis.

Plasma assays: Human leptin was measured using a commercially availableradioimmunoassay (RIA) (Linco Research, USA). All other plasma hormonelevels were measured using established in-house RIAs (Tarling et al.,Intensive Care Med. 23, 256-260, 1997). Glucose concentrations weremeasured using a YSI 2300STAT analyser (Yellow Springs Instruments Inc.,Ohio, USA). Plasma paracetamol levels were measured using an enzymaticcolorimetric assay (Olympus AU600 analyzer).

Human Studies: PYY₃₋₃₆ was purchased from Bachem (California, USA). TheLimulus Amoebocyte Lysate assay test for pyrogen was negative and thepeptide was sterile on culture. Ethical approval was obtained from theLocal Research Ethics Committee (project registration 2001/6094) and thestudy was performed in accordance with the principles of the Declarationof Helsinki. Subjects gave informed written consent.

Each subject was studied on two occasions with at least 1 week betweeneach study. Volunteers filled out a food diary for three days prior toeach infusion, and for the following 24 hours. All subjects fasted anddrank only water from 20:00 on the evening prior to each study. Subjectsarrived at 08:30 on each study day, were cannulated and then allowed torelax for 30 minutes prior to the onset of the study protocol. Bloodsamples were collected every 30 minutes into heparinised tubescontaining 5,000 Kallikrein Inhibitor Units (0.2 ml) of aprotinin(Bayer) and centrifuged. Plasma was separated and then stored at −70° C.until analysis. Subjects were infused with either saline or 0.8pmol·kg¹·min⁻¹PYY₃₋₃₆ for 90 minutes (about 72 pmol total infusion), ina double blind randomized crossover design.

Two hours after the termination of the infusion, subjects were offeredan excess free-choice buffet meal (Edwards et al., Am. J. Physiol.Endocrinol. Metab. 281, E155-E166, 2001), such that all appetites couldbe satisfied. Food and water were weighed pre- and postprandially andcaloric intake calculated. Appetite ratings were made on 100 mm visualanalogue scores (VAS) with the text expressing the most positive and thenegative rating anchored at each end (Raben et al., Br. J. Nutr. 73,517-30, 1995). VAS was used to assess hunger, satiety, fullness,prospective food consumption and nausea. Caloric intake following salineand PYY₃₋₃₆ were compared using a paired t test. The postprandialresponse curves were compared by ANOVA using repeated paired measures,with time and treatment as factors.

Measurements of Energy Expenditure: To determine the actions of PYY onenergy expenditure the OXYMAX system is utilized with rodents followingPYY injection into a treatment cohort. This system is also utilized withrodents following a saline injection (control cohort). The equipmentmeasures O₂ consumption and CO₂ production; the efficiency with whichthe body produces CO₂ from O₂ gives a reliable index of caloric ormetabolic efficiency. A similar system is used with human volunteers.

Example 2 Neural Network in the Arcuate Nucleus

A strain of transgenic mice was generated expressing green fluorescentprotein (EGFP Clontech), under the transcriptional control of mouse Pomcgenomic sequences that include a region located between −13 kb and −2 kbrequired for accurate neuronal expression (Young et al., J Neurosci 18,6631-40, 1998) (FIG. 1 a). Bright green fluorescence (509 nm) was seenin the two CNS regions where POMC is produced: the ARC and the nucleusof the solitary tract. Under ultraviolet (450-480 nm) excitation POMCneurons were clearly distinguished from adjacent, non-fluorescentneurons (FIG. 1 b) visualized under infrared optics. Doubleimmunofluorescence revealed >99% cellular co-localization of EGFP andPOMC peptides within the ARC (FIG. 1 c). There was close apposition ofboth tyrosine hydroxylase (TH)- and NPY-stained terminals onEGFP-expressing POMC neurons, but no evidence of co-localization of theTH or NPY immunoreactivity with EGFP. Total fluorescent cell countsperformed on coronal hypothalamic sections revealed 3148±62 (mean±SEM:n=3) POMC-EGFP neurons distributed through the entire ARC (Franklin etal., The Mouse Brain in Stereotaxic Coordinates, Academic Press, SanDiego, 1997) (FIG. 1 d). POMC neurons in the mouse are located bothmedially and ventrally within the ARC, in contrast to a predominantlylateral position in the rat ARC.

POMC-EGFP neurons in hypothalamic slices had a resting membranepotential of −40 to −45 mV and exhibited frequent spontaneous actionpotentials. The non-selective opioid agonist met-enkephalin (Met-Enk: 30μM; Sigma) caused a rapid (35-40 s), reversible hyperpolarization (10-20mV) of the membrane potential of POMC cells (n=10) and preventedspontaneous action potential generation (FIG. 2 a). In normal (2.5 mMK⁺) Krebs buffer, the reversal-potential of the inwardly-rectifyingopioid current was approximately −90 mV, while in 6.5 mM K⁺ Krebs thereversal-potential was shifted to approximately −60 mV (n=3: FIG. 2 b).The μ opioid receptor (MOP-R) antagonist CTAP (1 μM; PhoenixPharmaceuticals) completely prevented the current induced by Met-Enk inPOMC cells (n=3: FIG. 2 c). These characteristics indicate the opioidcurrent was due to activation of MOP-R and increased ion conductancethrough G protein coupled, inwardly-rectifying potassium channels (GIRK)(Kelly et al., Neuroendocrinology 52, 268-75, 1990). The similar opioidresponses in EGFP-labeled POMC neurons to that of a guinea pig (Kelly etal., Neuroendocrinology 52, 268-75, 1990) or mouse (Slugg et al.,Neuroendocrinology 72, 208-17, 2000). POMC cells, identified bypost-recording immunohistochemistry, suggests that expression of theEGFP transgene does not compromise either expression of receptors northeir coupling to second messenger systems in POMC neurons.

Next, the direct effects of leptin on identified POMC cells in slicepreparations were investigated. Leptin (0.1-100 nM) depolarized 72 of 77POMC cells by 3-30 mV (FIG. 3 a; mean±SEM depolarization at 100 nMleptin=9.7±1.2 mV, n=45) within 2-10 minutes, in a concentrationresponsive manner (FIG. 3 b). There were two components to thedepolarization and neither were fully reversible within 40 minutes.Firstly, the depolarization was due to a small inward current whichreversed at approximately −20 mV (FIG. 3 c), suggesting the involvementof a non-specific cation channel (Powis et al., Am J Physiol 274,R1468-72, 1998). Secondly, leptin treatment decreased the GABAergic toneonto POMC cells. GABAergic inhibitory postsynaptic currents (IPSCs) wereobserved in POMC cells and leptin (100 nM) decreased their frequency by25% (FIG. 3 d) in 5 out of 15 cells suggesting that it actedpresynaptically to reduce GABA release (leptin had no effect on IPSCs in10 out of 15 POMC neurons). The effect on IPSC frequency occurred with asimilar lag to the effect on membrane potential. Thus, leptin not onlydirectly depolarizes POMC neurons but also acts at GABAergic nerveterminals to reduce the release of GABA onto POMC neurons, allowing themto adopt a more depolarized resting potential. The consistentdepolarization of POMC cells by leptin was specific because leptin hadno effect on 5 of 13 adjacent non-fluorescent cells tested (FIG. 3 e),while it hyperpolarized 5 (FIG. 3 f) and depolarized 3 other non-POMCneurons in the ARC. The electrophysiological effects of leptin reportedhere are consistent with leptin's biological actions; leptin rapidlycauses release of α-MSH from rat hypothalami (Kim et al., J Clin Invest105, 1005-11, 2000), presumably by activating POMC neurons.

Previous reports of neuronal hyperpolarization by leptin (Glaum et al.,Mol Pharmacol 50, 230-5, 1996; Spanswick et al., Nature 390, 521-5,1997), and the demonstrated co-localization of GABA and NPY (Horvath etal., Brain Res 756, 283-6, 1997) within subpopulations of ARC neurons,led us to speculate that leptin hyperpolarizes NPY/GABA cells thatdirectly innervate POMC neurons, and thus reduces GABAergic drive ontoPOMC cells. Both the leptin and NPY Y2 receptors are expressed on NPYneurons in the ARC (Hakansson et al., J Neurosci 18, 559-72, 1998;Broberger et al., Neuroendocrinology 66, 393-408, 1997). Furthermore,activation of Y2 receptors inhibits NPY release from NPY neurons (Kinget al., J Neurochem 73, 641-6, 1999), and presumably would also diminishGABA release from NPY/GABA terminals. This is an alternativepharmacological approach, independent of leptin, to test thehypothesized innervation of POMC neurons by GABAergic NPY neurons.Indeed, NPY (100 nM; Bachem) decreased the frequency of GABAergic IPSCsby 55% within 3 minutes, in all 12 POMC cells tested (FIG. 4 a). BothNPY and leptin still inhibited IPSCs in the presence of tetrodotoxin(TTX) (6 of 6 and 3 of 5 cells respectively), indicating that some ofthe inhibition of IPSCs was occurring through direct effects atpresynaptic nerve terminals. POMC neurons express the NPY Y1 receptor(Broberger et al., Neuroendocrinology 66, 393-408, 1997) and NPY alsohyperpolarized all POMC neurons tested, by an average of 9±6 mV (n=3).

Another pharmacological test to confirm the origin of GABAergicinnervation on POMC neurons from NPY/GABA terminals was to test theeffect of the recently characterized and highly selective MC3-R agonistD-Trp⁸-γMSH (Grieco et al., J Med Chem 43, 4998-5002, 2000) on localGABA release. D-Trp⁸-γMSH (7 nM) increased the frequency of GABAergicIPSCs (280±90%) recorded from 3 of 4 POMC neurons (FIG. 4 b). It had noeffect on one cell. The positive effect of MC3-R activation, togetherwith the negative effects of NPY and leptin, demonstrate the dynamicrange of the NPY/GABA synapse onto POMC neurons and point to theimportant role of this synapse in modulating signal flow within the ARC.D-Trp⁸-γMSH (7 nM) also hyperpolarized (−5.5±2.4 mV) 9 of 15 POMCneurons tested and decreased the frequency of action potentials (FIG. 4c); the remaining cells showed no significant response to D-Trp⁸-γMSH.These effects could be due entirely to increased GABA release onto thePOMC cells, or could be due to an additional postsynaptic action ofD-Trp⁸-γMSH on POMC neurons, approximately half of which also expressthe MC3-R (Bagnol et al., J Neurosci (Online) 19, RC26, 1999). Thus,MC3-R acts in a similar autoreceptor manner to MOP-Rs on POMC neurons,diminishing POMC neuronal activity in response to elevated POMCpeptides.

To further determine that the IPSCs in POMC neurons were due to localinnervation by NPY/GABA cells, multi-label immunohistochemistry wasperformed using light and electron microscopy. Although independent NPY(Csiffary et al., Brain Res 506, 215-22, 1990) and GABA (Horvath et al.,Neuroscience 51, 391-9, 1992) innervation of POMC cells has beenreported, co-localization of NPY and GABA in nerve terminals formingsynapses onto POMC cells has not been shown. Similar to the rat(Csiffary et al., Brain Res 506, 215-22, 1990), a dense innervation ofPOMC cells by NPY axon terminals was detected in the mouse (FIG. 4 d).Electron microscopy confirmed the coexpression of NPY and GABA in axonterminals and revealed that these boutons established synapses on theperikarya of all 15 ARC POMC neurons analyzed (representative example,FIG. 4 e).

A detailed model of regulation of this circuit shows dual mechanisms ofleptin action in the ARC, interactions between NPY/GABA and POMCneurons, and autoregulatory feedback from opioid and melanocortinpeptides as well as NPY (FIG. 4 f). In this model, leptin directlydepolarizes the POMC neurons and simultaneously hyperpolarizes thesomata of NPY/GABA neurons, and diminishes release from NPY/GABAterminals. This diminished GABA release disinhibits the POMC neurons,and result in an activation of POMC neurons and an increased frequencyof action potentials.

Example 3 Administration of PYY Inhibits Food Intake

The orexigenic NPY and the anorectic alpha melanocortin stimulatinghormone (α-MSH) systems of the hypothalamic arcuate nucleus are involvedin the central regulation of appetite (Schwartz et al., Nature 404,661-671, 2000). However the potential mechanisms signaling mealingestion directly to these hypothalamic-feeding circuits are unclear.PYY₃₋₃₆ is a gut-derived hormone that is released postprandially inproportion to the calories ingested (Pedersen-Bjergaard et al., Scand.J. Clin. Lab. Invest. 56, 497-503, 1996). The effects of peripheraladministration of PYY₃₋₃₆ on feeding were investigated.

An intraperitoneal injection (IP) of PYY₃₋₃₆ to freely feeding rats,prior to the onset of the dark-phase, significantly decreased subsequentfood intake (FIG. 5 a). A similar inhibition of feeding was seenfollowing IP injection in rats fasted for 24 hours (FIG. 5 b). A timecourse of the plasma PYY₃₋₃₆ levels achieved following IP injection ofPYY₃₋₃₆ demonstrated a peak level at 15 minutes post injection, whichwas within the normal postprandial range (peak PYY₃₋₃₆ levels 15 minutespost IP injection of 0.3 μg/100 g=99.3±10.4 pmol/l vs. peak postprandiallevel=112.1±7.8 pmol/l, n=8-10 per group), suggesting that physiologicalconcentrations of PYY₃₋₃₆ inhibit feeding. PYY₃₋₃₆ did not affectgastric emptying (percentage of food ingested remaining in the stomachat 3 hours: PYY₃₋₃₆=36±1.9%, saline=37.4±1.0% n=12) (Barrachina et al.,Am. J. Physiol. 272, R1007-11, 1997). PYY₃₋₃₆ administered IP twicedaily for 7 days reduced cumulative food intake (7-day cumulative foodintake: PYY₃₋₃₆=187.6±2.7 g vs. saline=206.8±2.3, n=8 per group,P<0.0001) and decreased body weight gain (FIG. 5 d) (PYY₃₋₃₆=48.2±1.3 gvs. saline=58.7±1.9, n=8 per group, P<0.002).

Example 4 PYY Administration Affects c-fos Expression

To investigate whether this inhibition of food intake involved ahypothalamic pathway, c-fos expression was examined in the arcuatenucleus, an important center of feeding control (Schwartz et al., Nature404, 661-671, 2000; Cowley et al., Nature 411, 480-484, 2001), followinga single IP injection of PYY₃₋₃₆. There was a 2-fold increase in thenumber of cells positive for c-fos in the lateral arcuate of the rat(PYY₃₋₃₆=168±2, saline=82.7±5, n=3, P<0.0001). Likewise inPomc-EGFP-transgenic mice (Cowley et al., Nature 411, 480-484, 2001) IPadministration of PYY₃₋₃₆ resulted in a 1.8-fold increase in the numberof arcuate cells positive for c-fos (FIG. 6 b), compared with salinecontrol animals (FIG. 6 a) (PYY₃₋₃₆=250±40, saline=137±15, n=5, P<0.05).IP PYY₃₋₃₆ caused a 2.6 fold increase in the proportion of POMC neuronsthat express c-fos (PYY₃₋₃₆=20.4±2.9%, saline=8±1.4%, n=5, P<0.006)(FIGS. 6 c and d).

These observations suggested that PYY₃₋₃₆ may act via the arcuatenucleus. Thus, the actions of PYY₃₋₃₆, and its effects upon NPY and POMCcircuits in the hypothalamus, were studied. In view of the sustainedinhibition of food intake and the effects on weight gain followingperipheral administration of PYY₃₋₃₆ both Pomc and Npy hypothalamicmessenger RNA (mRNA) were measured using RNase protection assays. Asignificant decrease in Npy mRNA in response to PYY₃₋₃₆ was observed 6hours post IP injection, compared with saline treated animals(saline=17.3±2.0, PYY₃₋₃₆=8.8±1.0, relative optical density units,P<0.02). A non-significant increase occurred in Pomc mRNA levels.

Example 5 Y2 Receptors

PYY₃₋₃₆ shows a 70% amino acid sequence identity to NPY and acts throughNPY receptors (Soderberg et al., J. Neurochem. 75, 908-18, 2000). TheY2R is a putative inhibitory presynaptic receptor and is highlyexpressed on the arcuate NPY neurons (Broberger et al.,Neuroendocrinology 66, 393-408, 1997), though not on the neighboringPOMC neurons. PYY₃₋₃₆ is a high affinity agonist at the Y2 receptor(Grandt et al., Regul. Pept. 51, 151-159, 1994). It was hypothesizedthat peripheral PYY₃₋₃₆ inhibits food intake via the Y2R in the arcuatenucleus, an area known to be directly accessible to circulating hormones(Kalra et al., Endocr. Rev. 20, 68-100, 1999).

To investigate this hypothesis, PYY₃₋₃₆ was injected directly into thearcuate nucleus (Kim et al., Diabetes 49, 177-82, 2000). In rats fastedfor 24 hours, food intake was significantly decreased by doses as low as100 fmol (FIG. 7 a), resulting in a similar inhibition to that seenfollowing IP administration. To establish whether these effects were viathe Y2R, aY2R selective agonist was used (Potter et al., Eur. J.Pharmacol. 267, 253-262, 1994), N-acetyl (Leu²⁸, Leu³¹) NPY (24-36)[Y2A]. Its affinity was confirmed using receptor-binding studies (Smallet al., Proc. Natl. Acad. Sci. U.S.A. 94, 11686-91, 1997) on cell linesexpressing the NPY Y1, Y2 and Y5 receptors (Y2 IC₅₀=1.3±0.2 nM, Y1IC₅₀>5000 nM, Y5 IC₅₀>5000 nM). Intra-arcuate nucleus injection of Y2Ain rats previously fasted for 24 hours dose-dependently (100 fmol-1nmol) inhibited food intake (chow ingested 2 hours post-injection, 0.1nmol Y2A=6.2±0.5 g, saline=8.2±0.6 g, n=8 per group, P<0.05).

To confirm the anatomical specificity of this effect Y2A (100 fmol-1nmol) was injected into the paraventricular nucleus (PVN) (Kim et al.,J. Clin. Invest. 105, 1005-11, 2000) of rats fasted for 24 hours andfound no alteration of food intake (2 hour post-injection saline=8.3±0.4g, 0.1 nmol Y2A=8.0±0.6 g, n=8 per group). To further determine the roleof the Y2R in the feeding inhibition caused by peripheral PYY₃₋₃₆, theeffect of PYY₃₋₃₆ on Y2r-null mice and littermate controls was examined.PYY₃₋₃₆ inhibited daytime feeding in a dose responsive manner in fastedmale wild-type mice but did not inhibit food intake in fasted maleY2r-null mice (FIGS. 7 b and 7 c). Food intake measured in response to afast demonstrated that male Y2r-null mice eat significantly more at 2, 4and 24 hours compared with their littermate controls (24-hour cumulativefood intake; Y2r-null mice=7.1±0.48 g vs. wild-type=5.3±0.7 g, n=8 pergroup, P<0.05).

The electrophysiological response of hypothalamic POMC neurons toadministration of both PYY₃₋₃₆ and Y2A was examined. These neurons wereidentified using mice with targeted expression of green fluorescentprotein in POMC neurons (Cowley et al., Nature 411, 480-484, 2001).PYY₃₋₃₆ disinhibited the POMC neurons, resulting in a significantdepolarization of 19 of the 22 POMC neurons tested (FIG. 8 a inset)(10.3±2.1 mV depolarization, n=22, P<0.0003). A similar depolarizationwas seen with Y2A (8.7±1.8 mV depolarization, n=9, P<0.002). Thedepolarization caused by PYY₃₋₃₆ stimulated a significant increase inthe frequency of action potentials in POMC neurons (FIG. 8 a) (93%increase over control, P<0.05, n=22). In the whole cell mode the effectof PYY₃₋₃₆ was sometimes reversed upon washout, but only after a longlatency (30 minutes). A similar washout of leptin effects upon theseneurons was observed.

To exclude effects of cellular rundown, or seal deterioration, theeffects of PYY₃₋₃₆ in the “loose cell-attached” (or extracellular)configuration was examined. PYY₃₋₃₆ caused a reversible 5-fold increasein the frequency of action potentials in loose cell-attached recordingsof POMC neurons (FIG. 8 b). This increase in firing rate occurred withthe same latency as PYY₃₋₃₆ reduced the frequency of inhibitorypostsynaptic currents (IPSCs) onto all 13 POMC neurons tested (FIG. 8 c)(51.9±9.2% reduction, n=13, P<0.0001), indicating a reduced frequency ofGABA release onto POMC neurons. Interestingly, the firing rate of POMCneurons returned to basal, in spite of continued inhibition of IPSCs. Asimilar effect upon IPSC frequency was seen with Y2A (44.4±9.3%reduction, n=8, P<0.004) suggesting this effect to be via Y2R. PYY₃₋₃₆(25 nM) caused a hyperpolarization (5.2±1.16 mV, P<0.004, n=5) ofunidentified, but presumably NPY-containing, non-POMC, neurons in thearcuate nucleus. There is a tonic GABAergic inhibition of POMC neuronsby NPY neurons (Cowley et al., Nature 411, 480-484, 2001) and theseresults suggest that PYY₃₋₃₆ acts by inhibiting NPY neurons, thusdecreasing this GABAergic tone and consequentially disinhibiting POMCneurons. The effect of Y2A on peptide secretion was also examined usinghypothalamic explants (Kim et al., J. Clin. Invest. 105, 1005-11, 2000).Y2A significantly decreased NPY release, with a concomitant increase inα-MSH release from hypothalamic explants (FIGS. 8 d and 4 e). Takentogether, these observations suggest that PYY₃₋₃₆ modulates both the NPYand melanocortin systems in the arcuate nucleus.

Example 6 Human Studies

Because of the importance of the melanocortin system in man (Barsh etal., Nature 404, 644-651, 2000) and the profound effects of PYY₃₋₃₆ onboth feeding and weight change seen in rodents, the effects of PYY₃₋₃₆on appetite and food intake were investigated in human subjects. Twelvehealthy fasted, non-obese volunteers (six men and six women, mean age26.7±0.7 years, BMI=24.6±0.94 kg·m⁻²) were infused with PYY₃₋₃₆ (0.8pmol·kg⁻¹·min⁻¹) or saline for 90 minutes in a double-blind placebocontrolled crossover study.

PYY₃₋₃₆ plasma concentrations increased from mean basal concentration of8.3±1.0 pM to 43.5±3 pM during the PYY₃₋₃₆ infusion and mimickedpostprandial levels (Pedersen-Bjergaard et al., Scand. J. Clin. Lab.Invest. 56-, 497-503, 1996; Adrian et al., Gastroenterology 89,1070-1077, 1985). Post-infusion, PYY₃₋₃₆ concentrations returned tobasal within 30 minutes. PYY₃₋₃₆ infusion resulted in a significantdecrease in hunger scores (Raben et al., Br. J. Nutr. 73, 517-30, 1995)(FIG. 9 c), but not in the scores for sleepiness or sickness. Calorieintake during a free-choice buffet meal (Tarling et al., Intensive CareMed. 23, 256-260, 1997) two hours after the termination of the infusionwas reduced by over a third compared to saline (36±7.4%, p<0.0001) (FIG.9 a). There was no effect upon fluid intake and no difference insensations of fullness or nausea reported by the volunteers. PYY₃₋₃₆administration had no effect on gastric emptying, as estimated by theparacetamol absorption method (Edwards et al., Am. J. Physiol.Endocrinol. Metab. 281, E155-E166, 2001; Tarling et al., Intensive CareMed. 23, 256-260, 1997), or on plasma glucose, plasma leptin, GLP-1, orinsulin. Analysis of the food diaries revealed a significant inhibitionof food intake in the 12-hour period following the PYY₃₋₃₆ infusion(saline=2205±243 kcal, PYY₃₋₃₆=1474±207 kcal). However, food intakeduring a 12 to 24 hour period between the two groups was virtuallyidentical. Overall there was a 33% decrease in cumulative total calorieconsumption in the 24-hour period following the PYY₃₋₃₆ infusion (FIG. 9b). These findings demonstrate that infusion of PYY₃₋₃₆, matchingpostprandial levels, caused a marked inhibition of both appetite andfood intake in man.

In an additional study, two groups of healthy subjects (n=12 per group,6 males and 6 females), one with increased Body Mass Index (BMI)(mean=32.73+/−0.93 kg/m2) and another group with low BMI(mean=20.49+/−2.05 kg/m2), were studied on two occasions with at least 1week between each study. All subjects fasted and drank only water from20:00 hours on the evening prior to each study. Subjects arrived at08:30 on each study day, were cannulated and then allowed to relax for30 minutes prior to the onset of the study protocol. Subjects wereinfused with either saline or 0.8 pmol·kgl·min-1 PYY₃₋₃₆ for 90 minutes,in a double blind randomized crossover design. Two hours after thetermination of the infusion, subjects were offered an excess free-choicebuffet meal, such that all appetites could be satisfied. Food and waterwere weighed pre- and postprandially and caloric intake calculated.Caloric intake following saline and PYY₃₋₃₆ were compared using a pairedt test (p<0.001). The number of calories ingested followingadministration of PYY₃₋₃₆ differed significantly from the number ofcalories ingested following administration of saline for both theoverweight group and the lean group. The overweight group showed a28.8+/−4.3% reduction and the lean group a 31.1+/−4.4% reduction.However, the reduction for the overweight group did not differsignificantly from the reduction for the lean group. These findingsdemonstrate that infusion of PYY₃₋₃₆, matching postprandial levels,caused a marked inhibition of both appetite and food intake in both leanand overweight subjects.

Administration of PYY₃₋₃₆ at doses of 10 nmoles and 20 nmoles (insaline) subcutaneously to human subjects resulted in raised plasmalevels of PYY₃₋₃₆ as shown in FIG. 10.

Without being bound by theory, cells within the arcuate nucleus coulddetect circulating peripheral satiety signals and relay these signals toother brain regions (Butler et al., Nature Neuroscience 4, 605-611,2001). This is supported by the observation that leptin modifies theactivity of both the POMC and NPY arcuate neurons (Cowley et al., Nature411, 480-484, 2001). The results disclosed herein demonstrate, through acombination of electrophysiological and hypothalamic explant studies,that the gut hormone, PYY₃₋₃₆, can directly influence hypothalamiccircuits, resulting in coordinate changes in POMC and NPY action. Theresults presented here demonstrate that NPY neurons in the ARC are notprotected by the blood/brain barrier, and thus are accessible tocirculating molecules. Furthermore, PYY₃₋₃₆ administered directly intothis brain region reduces food intake.

The data disclosed herein demonstrates that postprandial levels ofPYY₃₋₃₆ inhibit food intake in more than one mammalian species (e.g.rodents and human subjects) for up to 12 hours, thereby demonstrating arole in regulation of food intake. This role can be described as a longterm role, such as over a period of several hours (e.g. at least two,three, four, eight, or twelve hours, or from about two to about fifteenhours). This is in contrast to previously characterized gut-derived‘short-term’ satiety signals, e.g. cholecystokinin (Schwartz et al.,Nature 404, 661-671, 2000; Moran, Nutrition 16, 858-865, 2000), theeffects of which are relatively short-lived (e.g., from about 1-4hours).

The failure of PYY₃₋₃₆ to inhibit food intake in the Y2r-null miceprovides evidence that PYY₃₋₃₆ reduces food intake via a Y2R dependentmechanism. The results disclosed herein suggest the existence of a novelgut-hypothalamic pathway in the regulation of feeding, involvingpostprandial PYY₃₋₃₆ acting at the arcuate Y2R. Thus, PYY, and analogsthereof, such as PYY₃₋₃₆ provide novel therapeutic agents for thetreatment of obesity.

Example 7 Parenterally Administered GLP-1

GLP-1 i.e. GLP-1 (7-36) amide (25 nmol/kg) or saline (control) wasadministered parenterally to non-fasted rats prior to the onset of thedark phase. Exendin 9-39 (20 nmoles/kg) or saline (control) injectedinto the arcuate nucleus of non-fasted rats. Food intake one hour laterwas measured.

The results as shown in FIG. 11 demonstrate that the effect on 1-hourfood intake in non-fasted rats of IP administration of GLP-1 is notinfluenced by the presence of concomitant exendin 9-39, a GLP-1 receptoragonist, in the arcuate nucleus of the rat. This indicates thatcirculating GLP-1 acts via the brainstem rather than the arcuatenucleus. These and other findings suggest that the main site forappetite inhibition by peripheral GLP-1 is the dorsal vagal complex,acting in part directly through the area postrema.

Example 8 Co-Administration of PYY₃₋₃₆ and GLP-1 in Rats

PYY₃₋₃₆ and GLP-1 i.e. GLP-1 (7-36) amide were injected(intraperitoneally) into non-fasted rats prior to the onset of the darkphase. GLP-1 and PYY₃₋₃₆ were administered separately at the followingdoses: GLP-1 30 μg/kg=1×G or 60 μg/kg=2×G in 500 μl saline; PYY₃₋₃₆ (3μg/kg=1×P or 6 μg/kg=2×P in 500 μl saline). PYY₃₋₃₆ and GLP-1 wereco-administrated IP (P+G group) at doses of GLP-1 30 μg/kg and PYY₃₋₃₆ 3μg/kg combined.

The results set out in FIG. 12 show that there is a clear synergisticeffect on the reduction of food intake when PYY₃₋₃₆ and GLP-1 wereco-administered (P+G). However, when the procedure was carried outsubstituting oxyntomodulin for GLP-1, the results obtained for reductionof food intake when PYY₃₋₃₆ and oxynotomdulin were co-administered wereonly the sum of the individual effects. No synergistic effect wasobserved.

Example 9 Co-Administration of PYY₃₋₃₆ and GLP-1 in Humans

10 healthy volunteers (4 men and 6 women) having a mean age of 24.7(+/−0.8) years and a mean BMI of 22.7 (+/−0.6) kg/m² were recruited. Thevolunteers were asked to fast and to drink only water from 9 pm on thenight preceding all study days. Each volunteer attended for four studydays. The volunteers arrived at 9.30 am and the venflons were inserted.A rest period from 10.00 to 10.30 followed. Intravenous infusion startedat 10.30, and a buffet lunch was provided between 12.00 and 12.30.Infusion was terminated at 12.30, and the volunteers went home at 13.30.

The volunteers were given the following intra-venous infusions for 120minutes (90 minutes pre-meal) in random order: Saline, GLP-1, PYY₃₋₃₆,PYY₃₋₃₆+GLP-1. The dose of both GLP-1 and PYY3-36 was 0.4 pmol/kg/min.The study was double blinded. GLP-1 is i.e. GLP-1 (7-36) amide

The results of the food intake from the free buffet meal are given inthe Table below

Food Intake from Free Buffet Meal (Kcal)

Saline G P P + G mean 997 950 848 724 Sem 68.0 81.3 105.2 66.6 p vsaline 0.336318995 0.069375886 3.00605E−06 p v P + G 0.0036225230.044418337 % reduction 5% 15% 27% from saline

Using Paired t Tests for all Statistical Comparisons

These result demonstrate a synergistic effect when PYY3-36 and GLP-1 areco-administered.

The results obtained in Examples 4 and 5 above demonstrate that PYY₃₋₃₆acts via the arcuate nucleus. Oxynotmodulin is also considered to actvia that region of the brain. The results obtained in Example 8 aboveindicates that GLP-1 acts via the brainstem. It appears from the resultsobtained in this Example that, when agents such as PYY₃₋₃₆ and GLP-1that act via different areas of the brain and/or by differentneurological routes are administered parenterally, they produce asynergistic affect, whereas when they act by the same area and/or routesuch as PYY₃₋₃₆ and oxynotmodulin, they do not. The synergistic effecton food intake obtained when PYY and GLP-1 were co-administered tohumans confirms the observation made in rats.

These results indicate the importance in vivo of the interaction of guthormones on the regulation of appetite and food intake. The presentinvention demonstrates that, surprisingly, it is possible to regulateappetite and food intake more effectively by informed choice of agents.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described disclosure. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. A method for decreasing calorie intake or food intake in a subject,for preventing or reducing weight gain or inducing weight loss in asubject, or controlling any one or more of appetite, satiety, and hungerin a subject comprising peripherally administering a therapeuticallyeffective amount of Peptide YY (PYY) or an agonist thereof and atherapeutically effective amount of Glucagon-like peptide-1 (GLP-1) oran agonist thereof to the subject, wherein the amount of PYY or agonistthereof is administered in a molar equivalent amount of a PYY₃₋₃₆ doseof no more than 2.2 nmol per kg body weight of the subject. 2-8.(canceled)
 9. A method as claimed in claim 1, wherein the PYY or agonistthereof and the GLP-1 or agonist thereof are administered simultaneouslyor sequentially.
 10. A method as claimed in claim 1, wherein the PYY andthe GLP-1 are administered via different routes.
 11. A method as claimedin claim 1, wherein the subject is overweight, obese or diabetic. 12.(canceled)
 13. A method as claimed in claim 1, wherein the subject hastype II diabetes.
 14. A method as claimed in claim 1, wherein peripheraladministration comprises subcutaneous, intravenous, intramuscular,intranasal, transdermal or sublingual administration.
 15. A method asclaimed in claim 1, wherein peripherally administering PYY or theagonist thereof comprises administering about 45 to about 135 pmol perkilogram body weight of the subject or about 72 pmol per kilogram bodyweight of the subject.
 16. (canceled)
 17. A method as claimed in claim15, wherein peripherally administering PYY or the agonist thereofcomprises administering about 45 to about 135 pmol per kilogram bodyweight of the subject at least 30 minutes prior to a meal.
 18. A methodas claimed in claim 1, wherein peripherally administering thetherapeutically effective amount of PYY or the agonist thereof comprisesadministering PYY or an agonist thereof to the subject in a multitude ofdoses, wherein each dose in the multitude of doses comprisesadministration of about 0.5 to about 135 pmol per kilogram of bodyweight at least about 30 minutes prior to a meal.
 19. (canceled)
 20. Amethod as claimed in claim 1, wherein the PYY or the agonist thereof isadministered in an amount sufficient to decrease calorie intake for aperiod of about 2 to 12 hours.
 21. A method as claimed in claim 1,wherein the PYY or agonist thereof is administered peripherally at adose of 0.1 nmoles per kg body weight of the subject, 0.2 nmoles per kgbody weight of the subject, 0.4 nmoles per kg body weight of thesubject, 0.6 nmoles per kg body weight of the subject, 0.8 nmoles per kgbody weight of the subject, 1.0 nmole per kg body weight of the subject,1.2 nmoles per kg body weight of the subject, 1.4 nmoles per kg bodyweight of the subject, 1.6 nmoles per kg body weight of the subject, 1.8nmoles per kg body weight of the subject, 2.0 nmoles per kg body weightof the subject, or 2.2 nmoles per kg body weight of the subject. 22.(canceled)
 23. A method as claimed in claim 1, wherein the GLP-1 oragonist thereof is administered peripherally at a dose of 0.1 nmoles perkg body weight of the subject or more, for example, 0.2 nmoles or more,for example, 0.4 nmoles or more, for example, 0.6 nmoles or more, forexample, 0.8 nmoles or more, for example, 1.0 nmole or more, forexample, 1.2 nmoles or more, for example, 1.4 nmoles or more, forexample, 1.6 nmoles or more, for example, 1.8 nmoles or more, forexample, 2.0 nmoles or more, for example, 2.2 nmoles or more, forexample, 2.4 nmoles or more, for example, 2.6 nmoles or more, forexample, 2.8 nmoles, for example, 3.0 nmoles or more, for example, up to3.2 nmoles per kg body weight.
 24. (canceled)
 25. (canceled)
 26. Amethod as claimed in claim 1, wherein the PYY or agonist thereof ismodified by one or more of amidation, glycosylation, acylation,sulfation, phosphorylation, cyclization, lipidization, or pegylation.27. A method as claimed in claim 1, wherein the GLP-1 or agonist thereofcomprises the amino acid sequence of GLP-1 (7-36) (SEQ ID NO:339) or avariant thereof that binds a GLP-1 receptor.
 28. A method as claimed inclaim 1, wherein the PYY is PYY₃₋₃₆ or variant thereof.
 29. (canceled)30. (canceled)
 31. A method as claimed in claim 1, wherein the GLP-1agonist is exendin-4 or a derivative thereof that is a GLP-1 agonist.32. A method as claimed in claim 1, further comprising administering atherapeutically effective amount of amfepramone (diethylpropion),phentermine, mazindol, phenylpropanolamine, fenfluramine,dexfenfluramine, or fluoxetine.
 33. A method as claimed in claim 1,wherein the subject is human. 34-43. (canceled)
 44. A pharmaceuticalcomposition comprising PYY and an agonist thereof and GLP-1 or anagonist thereof, in admixture or in conjunction with a pharmaceuticallysuitable carrier, wherein the amount of PYY or agonist thereof presentin the composition is sufficient to deliver a molar equivalent amount ofPYY₃₋₃₆ of no more than 2.2 nmol per kg body weight of the subject. 45.A pharmaceutical composition comprising PYY or an agonist thereof inadmixture with a pharmaceutically suitable carrier, in a form suitablefor subcutaneous, intravenous, intramuscular, intranasal, transdermal,or sublingual administration, wherein the amount of PYY or agonistthereof present in the composition is sufficient to deliver a molarequivalent amount of PYY₃₋₃₆ of no more than 2.2 nmol per kg body weightof the subject.
 46. A pharmaceutical composition as claimed in claim 45,which comprises 20 nmoles or more, for example, 30 nmoles or more, forexample, 40 nmoles or more of PYY or an agonist thereof.
 47. (canceled)48. A pharmaceutical composition as claimed in claim 45, which comprisesfrom PYY or an agonist thereof in an amount suitable for administrationperipherally in an amount of up to 2.2 nmoles per kg body weight, forexample, up to 2.0 nmoles, for example, up to 1.8 nmoles, for example,up to 1.4 nmoles, for example, up to 1.2 nmoles, for example, up to 1.0nmoles, for example, up to 0.8 nmoles, for example, up to 0.6 nmoles,for example, up to 0.4 nmoles, for example, up to 0.2 nmoles, forexample, up to 0.1 nmoles per kg body weight.
 49. (canceled)
 50. Apharmaceutical composition as claimed in claim 48, in unit dosage form.51. A method as defined in claim 1, which comprises administering PYY oran agonist thereof subcutaneously at a dose of 10 nmoles or more, forexample, 20 nmoles or more, for example, 30 nmoles or more, for example,40 nmoles or more to the subject of the method. 52-57. (canceled)
 58. Amethod as claimed in claim 1, wherein the PYY or agonist thereof isadministered in an amount of from 1 to 100 nmols, from 2 to 90 nmols,from 5 to 80 nmols, or from 5 to 50 nmols.